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The Mechanism of Formyl-Coenzyme A Transferase, a Family III CoA Transferase, from Oxalobacter formigenes

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PAGE 1

THE MECHANISM OF FORMYL-COENZ YME A TRANSFERASE, A FAMILY III COA TRANSFERASE, FROM Oxalobacter formigenes By STEFN J"NSSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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This dissertation is dedicated to my family.

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iii ACKNOWLEDGMENTS This study was supported by grants from the National Institutes of Health (DK61666 and DK53556) and by the University of Florida Chemistry Department. Partial funding was also receiv ed from Dr. Ammon B. Peck. Thanks go to my doctoral dissertation committee: Dr. Steven A. Benner, Dr. Ronald K. Castellano, Dr. Ammon B. Peck, Dr. Jon D. Stewart, and especially my advisor, Dr. Nigel G. J. Richards, for making this research project a reality. Stefano Ricagno and Dr. Ylva Lindqvist, I thank for fruitf ul collaboration and their excellent crystallographic work. I am grateful to my coworkers and fri ends in the Richards research group, especially Dr. Christopher H. Chang for pr oofreading, countless discussions and valuable insights, Draenka Svedrui for her help, and Sue Abbatie llo for her mass spectrometry efforts, and Dr. Jianqiang Wang for s upplying thiocresol oxalate monoester. I thank Dr. James A. Deyrup, Graduate Coordi nator, and Lori Clark in the Graduate Student Office for their help and advice. Dr. Harmeet Sidhu, Ixion Biotechnology, Inc ., I thank for provi ding the original expression strain of E. coli training, and for use of equi pment and resources at Ixion during the summer of 2000. Finally, I thank my family for suppor ting me, especially my wife, slaug Hgnadttir, for all of her support and love throughout my graduate studies and for proofreading.

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iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Oxalic Acid Breakdown...............................................................................................1 Coenzyme A Transferases............................................................................................3 Overview...............................................................................................................3 Formyl-CoA Transferase.......................................................................................5 Kinetics of Bisubstrate Enzymes..................................................................................8 Overview...............................................................................................................8 Initial Velocity Studies..........................................................................................9 Product Inhibition Studies...................................................................................11 Research Objective.....................................................................................................13 2 EXPRESSION, PURIFICATION, AND KINETIC STUDIES OF FORMYL-COA TRANSFERASE........................................................................................................14 Expression and Purification of Reco mbinant Formyl-CoA Transferase....................14 Synthesis of CoA Esters.............................................................................................16 Formyl-CoA........................................................................................................16 Oxalyl-CoA.........................................................................................................17 Purification..........................................................................................................17 Stability................................................................................................................18 Assay Development....................................................................................................19 Equilibrium Constant..................................................................................................23 Alternative Substrates.................................................................................................23 Kinetics....................................................................................................................... 25 Initial Rates..........................................................................................................26 Product Inhibition................................................................................................28 Inhibition by formate....................................................................................29 Inhibition by oxalyl-CoA.............................................................................31

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v Inhibition by Coenzyme A..................................................................................32 3 STRUCTURE AND MECHANISM OF FORMYL-COA TRANSFERASE...........33 Structure...................................................................................................................... 33 Active Site..................................................................................................................36 Point Mutation Studies...............................................................................................39 Mechanism..................................................................................................................42 4 EXPERIMENTAL......................................................................................................47 Expression and Purification of FRC...........................................................................47 Expression of Selenomethionine De rivative of Wild-Type FRC...............................49 Site-Directed Mutagenesis..........................................................................................50 Synthesis of CoA Esters.............................................................................................50 Formyl-CoA........................................................................................................50 Oxalyl-CoA.........................................................................................................51 Analysis of CoA Esters.......................................................................................52 Purification of CoA Esters...................................................................................52 Enzymatic Assay........................................................................................................53 Equilibrium Constant Determination..........................................................................54 Size Exclusion Chromatography (SEC).....................................................................54 5 SUMMARY................................................................................................................55 APPENDIX A PROTEIN PURIFICATION CHROMATOGRAMS.................................................59 B NMR SPECTRA OF FORMYLTHIOPHENYL ESTER..........................................62 LIST OF REFERENCES...................................................................................................64 BIOGRAPHICAL SKETCH.............................................................................................68

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vi LIST OF TABLES Table page 1-1. Previously reported pr operties of native wild-type fo rmyl-CoA transferase from Oxalobacter formigenes .............................................................................................6 2-1. Purification table of FRC showing typical yiel d and purification level....................15 2-2. Effect of excessive dilution of the enzyme stock solution on specific activity.........22 2-3. Summary of alternativ e substrates screening............................................................24 2-4. Kinetic constants of FRC calculated from initial rates data (Figures 2-9 and 2-10).26 2-5. Product inhibition patte rns observed for various mechanisms of bisubstrate enzymes....................................................................................................................28 2-6. Inhibition constants for formate and oxalyl-CoA......................................................28

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vii LIST OF FIGURES Figure page 1-1. Currently recognized classes of enzyme s that catalyze the direct or indirect degradation of oxalate................................................................................................1 1-2. Structure of Coenzyme A............................................................................................4 1-3. Previously known enzyme-cataly zed mechanisms of CoA transfer...........................4 1-4. Crystal structure of the inte rlocked FRC dimer with bound CoA...............................7 1-5. Cleland notation of the three ma in types of Bi Bi mechanisms..................................9 1-6. Ordered Bi Bi mechanism showing the individual rate constants (top), and the equation for the initial forw ard velocity in the absen ce of products (bottom).........10 1-7. Lineweaver-Burk plots of initial ve locity data for a bisubstrate enzyme..................11 1-8. Lineweaver-Burk plots showing i nhibition patterns in enzyme kinetics..................12 2-1. SDS-PAGE gels showing the expr ession level and purif ication of FRC..................15 2-2. Synthetic schemes for formyl-CoA and oxalyl-CoA................................................16 2-3. Three HPLC chromatograms showi ng the hydrolysis of oxalyl-CoA by KOH........17 2-4. Three HPLC chromatograms showing ex traction of thiocresol oxalate monoester from oxalyl-CoA reaction mixture...........................................................................18 2-5. Graphs for determining the rate of hydrolysis of the CoA esters..............................19 2-6. HPLC chromatograms of assay mixtur e aliquots quenched after 5 min (A), 15 min (B), and 55 min (C)..................................................................................................20 2-7. Linear increase of initial rate with increasing enzyme concentration.......................22 2-8. Structures of free acids and ester parts of CoA esters used fo r alternative substrate screening (see Table 2-3).........................................................................................25 2-9. Lineweaver-Burk plot of initial rates at three fixed formyl-CoA concentrations.....27

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viii 2-10. Lineweaver-Burk plot of initial rate s at four fixed oxalate concentrations.............27 2-11. Lineweaver-Burk plot showing product inhibition by formate vs. formyl-CoA at fixed unsaturating oxalate concentration..................................................................29 2-12. Lineweaver-Burk plot showing product inhibition by formate vs. formyl-CoA at fixed saturating oxalate concentration......................................................................30 2-13. Lineweaver-Burk plot showing product inhibition by formate vs. oxalate at fixed unsaturating formyl-CoA concentration...................................................................30 2-14. Lineweaver-Burk plot showing produc t inhibition by oxalylCoA vs. oxalate at fixed unsaturating formyl-CoA concentration.........................................................31 2-15. Lineweaver-Burk plot s howing inhibition by CoA vs. oxalate at fixed unsaturating formyl-CoA concentration.......................................................................................32 3-1. Monomer and homodimer structures of recombinant formyl -CoA transferase........34 3-2. Size exclusion chromatography data used to calculate the molecular mass of FRC.35 3-3. Size exclusion HPLC chromatograms of FRC..........................................................35 3-4. Stereo picture of Coenzy me A in its binding site......................................................36 3-5. CLUSTAL W (1.82) multiple sequence alig nment of Family III CoA transferases.37 3-6. Stereo images of activ e site structures of FRC mu tants complexed with CoA.........40 3-7. Stereo picture of the e nd of the pantetheine chain of CoA and amino acids in the surrounding active site.............................................................................................42 3-8. Proposed mechanism of fo rmyl-CoA transferase from Oxalobacter formigenes .....43 3-9. Stereo picture showing the interacti ons of the oxalyl-aspartyl anhydride with residues in the FRC dimer........................................................................................45 A-1. FPLC chromatogram of the DEAE anion exchange purification step.....................59 A-2. FPLC chromatogram of the Bl ueFF affinity purification step.................................60 A-3. FPLC chromatogram of the buf fer exchange purification step................................60 A-4. FPLC chromatogram of the QHP anion exchange purification step........................61 B-1. 1H-NMR spectrum of formylthiophenyl ester..........................................................62 B-2. 13C-NMR spectrum of formylthiophenyl ester.........................................................63

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ix B-3. Close-up on relevant peaks of Figure A-2................................................................63

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE MECHANISM OF FORMYL-COENZ YME A TRANSFERASE, A FAMILY III COA TRANSFERASE, FROM Oxalobacter formigenes By Stefn Jnsson December 2004 Chair: Nigel G. J. Richards Major Department: Chemistry Formyl-Coenzyme A transferase (FRC) is a part of an oxalate -degrading catalytic cycle in Oxalobacter formigenes a bacterium which colonizes the gastrointestinal tract of many mammals, including humans, symbiotically degrading toxic oxalate ingested with food and produced as a byproduct of normal cellu lar metabolism. FRC is a member of a recently recognized Family III CoA transf erases, which apparently use a novel mechanism of CoA transfer as indicated by th e limited kinetic studies that have been published so far. FRC from O. formigenes was overexpressed in Escherichia coli and purified by anionic exchange and affinity chromatogra phy. The selenomethionine derivative of FRC was also expressed and purified, allowing dete rmination of the X-ray crystal structure of the enzyme. Mutations of the putative main cat alytic residue, identi fied by analysis of the crystal structure of FRC w ith bound CoA, caused very diminished or complete loss of transferase activity. Steady-state initial ra te kinetic studies on the wild-type enzyme

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xi indicate a ternary complex (sequential) mech anism rather than Ping-Pong kinetics, which are observed for the well known Family I CoA transferases, and product inhibition studies strongly support an ordered Bi Bi mechanism. A catalytic mechanism is proposed, based on the crystal structure and kine tic data, where the main catalytic residue forms mixed anhydrides with formate and oxalate during catalytic turnover. One of these proposed intermediates, an aspartyl-oxalyl anhy dride, was observed in a crystal structure obtained from crystals of the wild-type FRC grown in the presence of oxalyl-CoA, lending further evidence to the proposed mechanism.

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1 CHAPTER 1 INTRODUCTION Oxalic Acid Breakdown Oxalic acid is a byproduct of normal cellular metabolism and is toxic to almost all organisms.1 Several oxalate-degrading processe s have therefore naturally evolved (Figure 1-1): O O O O O O O O H O O SCoA O O O H O O O O O O O H SCoA O SCoA O O O H O O Oxalate oxidase O2 2H+(A) (B) cat. O2 H+Oxalate decarboxylase 2CO2 + H2O2CO2 + Oxalyl-CoA reductase (Glyoxylate dehydrogenase) (D) NAD(P)H NAD(P)++ CoAS-2 HO2C2O2 -2 [(HO2C2O2 -)Mn+3]+2O24 CO2H2O2 + 2 Mn+2Manganese peroxidase 2 Mn+32 H2O2 H+(C) (E) H++ Formyl-CoA transferase + Oxalyl-CoA decarboxylase CO2 Figure 1-1. Currently recognized classes of enzy mes that catalyze the direct or indirect degradation of oxalate in (A) plants and fungi, (B) fungi and bacteria, (C) fungi only, and (D, E) bacteria only.

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2 (A) Oxalate oxidase, f ound mainly in plants2 and more recently in fungi,3 catalyzes the oxidation of oxalate to carbon dioxide with concomitant reduction of dioxygen to hydrogen peroxide. (B) Oxalate decarboxylase found mainly in fungi4 and more recently in bacteria,5 decarboxylates oxalate in the presen ce of catalytic amounts of dioxygen. (C) Manganese peroxidase, secreted by so me fungi, catalyzes the formation of Mn+3 from Mn+2 and hydrogen peroxide. Oxalate-Mn+3 complexes then spontaneously form and break down to Mn+2, carbon dioxide and hydrogen peroxide in the presence of dioxygen.6-8 (D) Oxalyl-CoA reductase, also known as glyoxylate dehydrogenase, found in bacteria able to use oxalate as a source of carbon (oxalotrophic bacteria), catalyzes the NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dependent reduction of oxalyl-CoA to glyoxylate and free CoA.9,10 (E) Formyl-CoA transferase (FRC)11 and oxalyl-CoA decarboxylase (OXC),12,13 also found in oxalotrophic bacteria, form a catalytic cycle that breaks down oxalate to carbon dioxide and formate via formyland oxalyl-CoA ester intermediates. Bacterial enzymes are the least studied of th e oxalate-degrading enzymes. Of particular interest are FRC and OXC, which have been purified and characterized from Oxalobacter formigenes an anaerobic Gram-negative bacterium, which colonizes the gastrointestinal tract of many warm-blooded animals, including humans.11,13,14 O. formigenes is unique since oxalate is the only compound that s upports its growth, although small amounts of acetate are also required.14 Approximately 99% of the oxalate consumed by the bacterium is decarboxylated to CO2 and formate by OXC and FRC (Figure 1-1E) and the rest is used for cell biosynthesis, presumab ly through the action of oxalyl-CoA reductase (Figure 1-1D).14-17 OXC and FRC activities are ther efore crucial for the bacterium’s survival and play a central role in its oxala te metabolism. Absence of this bacterium from human intestinal flora has been strongl y linked to pathological conditions that can arise if oxalate accumulates in the human body, including hyperoxaluri a (increased levels of oxalate in urine), the formation of ki dney stones (urolithia sis), renal failure,

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3 cardiomyopathy, and cardiac conductance disorders.18-23 OXC is a TPP-dependent (thiamine pyrophosphate-dependent) decarboxylase.13 The mechanisms of TPPdependent enzymes are generally well known24 and will not be discussed here. The amino acid sequence of FRC, however, bears no similarity to the well known Family I CoA transferases or Family II CoA transfer ases. Instead, FRC, by sequence similarity, belongs to a newly recognized Family III CoA transferases (Pfam accession number PF02515),25 for which only limited kinetic studies11,26-31 and no mechanistic studies have been reported until now. Coenzyme A Transferases Overview The reversible transfer of the CoA moiety (Figure 1-2) from CoA thioesters to free acids is catalyzed by CoA transferases (Fi gure 1-1E) which were, until recently, grouped into two enzyme families. The well known Family I contains CoA transferases for 3oxoacids, short-chain fatty acids, and glutacona te. The transfer reaction proceeds via a Ping-Pong mechanism, where a glutamate resi due of the enzyme forms covalently bound anhydrides and CoA thioesters during catalysis.32 These enzymes incorporate 18O when 18O-containing CoA acceptor is used in the r eaction since one oxygen atom is transferred from the incoming free acid to the main ca talytic residue during one catalytic cycle (Figure 1-3A). Family I CoA transfer ases are inactivated by incubation with hydroxylamine or sodium borohydride (NaBH4) in the presence of CoA thioesters.32-36 Family II includes only the homodimeric -subunits of citrate lyase and citramalate lyase, which catalyze the transfer of an acyl carrier protein (ACP) c ontaining a covalently bound CoA derivative. In a ternary complex me chanism a direct attack of the incoming citrate or citramalate on the acetyl thioester of the acetylated CoA derivative results in the

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4 formation of a mixed anhydride of acetate a nd citrate or citramalate during catalytic turnover (Figure 1-3B). No covalent enzyme -substrate intermediates are formed in the mechanism.37-39 O H PO OH O OP O OH O PO OH O O N N N N NH2 OH N O H N SH O H OH } Adenine } Ribose-3-phosphate { { Pyrophosphate Pantetheine Figure 1-2. Structure of Coenzyme A. Glu O O R1 SCoA O Glu O O R1 O Glu SCoA O O R1 O R2 O O R1 O O Glu SCoA O R2 O O Glu O O R2 O Glu O O R2 SCoA O R2 SCoA O R1 SCoA O ACP-CoAS O R O O R O O O R SCoA-ACP O O O -SCoA -SCoA (A) (B) ACP-CoASFigure 1-3. Previously known enzyme-catalyz ed mechanisms of CoA transfer. (A) PingPong mechanism of Family I CoA tran sferases. Oxygen atoms from the incoming free acid are shown in bold f ont. (B) Ternary complex mechanism of Family II CoA transferases. The new Family III of CoA transferases cu rrently includes only three characterized enzymes other than formyl-CoA transferase from O. formigenes (FRC). These are BbsF

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5 (succinyl-CoA: ( R )-benzylsuccinate CoA transferase from Thauera aromatica ),26,27 FldA (( E )-cinnamoyl-CoA: ( R )-phenyllactate CoA transferas e from Stickland-fermenting Clostridia ),28,29 and CaiB (butyrobetainyl-CoA: ( R )-carnitine CoA transferase from E. coli and Proteus sp.).30,31 The subunits of these enzymes have similar masses, 42-47 kDa, while their quaternary structures include homodimers (FRC and CaiB), 22 aggregates (BbsF), and a subunit in a he terotrimeric enzyme complex (FldA). Additionally, there are many putative proteins that have been identified as probable Family III CoA transferases based on DNA and/or translated amino acid sequence similarities. A ternary mechanism with no covalent enzyme-substrate intermediates, similar to the mechanism of Family II CoA transferases, has been proposed for Family III enzymes based on the limited kinetic data repor ted that indicate a sequential mechanism rather than Ping-Pong.25 Formyl-CoA Transferase Unmodified native wild-type FRC has 428 amino acids. The monomeric mass is 47.3 kDa and its pI is 5.2 calculate d from the amino acid sequence.40 FRC catalyzes transfer of the CoA moiety from formyl -CoA to oxalate producing oxalyl-CoA and formate (Figure 1-1E). Previous literature on this enzyme is limited to earlier studies on the oxalate-degrading Pseudomonas oxalaticus ,9,12 which were the first indication of the existence of such an enzyme, and more recently the purification and limited characterization of FRC isolated from Oxalobacter formigenes ,11 the cloning of the FRC gene from O. formigenes and subsequent overexpression of the recombinant FRC in Escherichia coli .40 In addition, a gene ( yfdW ) from E. coli sharing 61% sequence identity with FRC has been cloned and expr essed as a His-tagged gene product.41 It is not yet known whether the yfdW gene product is an active transf erase. There are no reports of

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6 oxalate degradation by E. coli and it is not known if the yfdW gene is expressed under standard growth conditions or what factors could induce its expression. Table 1-1. Previously reporte d properties of native wild -type formyl-CoA transferase from Oxalobacter formigenes One unit (U) equals one micromole of CoA transferred per minute.11,40 Properties Values / Observations Km (formyl-CoA) vs. excess succinate 3.0 0.5 mM Vmax (formyl-CoA) vs. excess succinate 30 U/mg Km (oxalate) vs. formyl-CoA 5.1 0.5 mM Vmax (oxalate) vs. formyl-CoA 6.4 U/mg Km (succinate) vs. formyl-CoA 2.3 0.6 mM Vmax (succinate) vs. formyl-CoA 19 U/mg Maximum specific activity for transfer of CoA from formyl-CoA to oxalate 2.2 U/mg pH optimum 6.5 – 7.5 Isoelectric point (calculated) 5.2 Isoelectric point (experimental) 4.7 Molecular weight (calculated) 47.3 kDa Molecular weight (experimental ) 44.7 kDa (active as monomer) Acceptable CoA acceptors Oxalate, succinate Not acceptable CoA acceptors Acetate, malonate Acceptable CoA donors Formyl-CoA, succinyl-CoA Not acceptable CoA donors Acetyl-CoA Inhibition 20% by 1.0 mM N -ethylmaleimide 91% by 1.0 mM p -chloromercuribenzoate No inhibition 10 mM EDTA, 10 mM Ca+2, 10 mM Mg+2, 1.0 mM TPP In the previous characte rization the enzyme was assayed with a continuous, coupled enzyme assay in which formate dehydrogenase (FDH) was used to detect the rate of formate production by monitoring the formation of NADH (reduced form of nicotinamide adenine dinucleotide) spectropho tometrically. Findings of the previous characterization of the native wild-t ype FRC are summarized in Table 1-1.11 Although formyl-CoA and succinyl-CoA can both act as CoA donors, there is no report of oxalyl-

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7 CoA acting as one, so there is so far no ev idence of FRC being able to catalyze the reverse reaction (transfer of Co A from oxalyl-CoA to formate). Figure 1-4. Crystal structure of the interlocked FRC dimer with bound CoA. For clarity, the protein monomers are colored red and green and represented by molecular ribbons. The bound CoA molecules are s hown as space-filling models, which are colored using the following scheme: H white; C grey; N blue; O red; S yellow; P purple. The structure of recombinant FRC from O. formigenes has recently been published and represents the first crystal structure of a member of the new Family III CoA transferases.42 The structure is a novel fold wher e two circular shaped monomers are interlocked like two links in a chain creating a tightly pack ed homodimer. Each dimer has two active sites located on opposite sides of the structure in a cleft between the monomers (Figure 1-4). The structure, with bound coenzyme A molecules, allowed D169 to be identified as the most likely ma in catalytic residue (see Chapter 3). The yfdW gene from E. coli yields a protein with the same structure.41

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8 Kinetics of Bisubstrate Enzymes Overview The majority of enzymes catalyze reactio ns between two or more substrates yielding two or more products. Enzymes that use two substrates and yield two products, such as FRC, employ one of several possi ble Bi Bi mechanisms (using Cleland’s nomenclature where Bi Bi refers to two substrates and two products).43 The three main forms of Bi Bi mechanisms are (A) sequent ial random Bi Bi, wher e substrates bind in random order and products are released in ra ndom order, (B) sequential ordered Bi Bi, where substrates bind in an ordered manner a nd products are released in a specific order, and (C) Ping-Pong Bi Bi, where the first product is released before binding of the second substrate (Figure 1-5).43 The sequential mechanisms are also known as ternary complex mechanisms. Some variations of these general scenarios exist, but are not relevant to this discussion. Furthermore, the above systems can be under either rapid equilibrium conditions or steady-state conditions. Rapi d equilibrium conditions refer to when all binding and dissociation steps are very rapid compared to the catalytic step, and the rate-limiting step is the breakdown of EAB to E + P + Q. This describes some random binding systems well, but the steady-state a pproach is preferred for se quential ordered and Ping-Pong mechanisms.44 The steady-state approach describe s systems where the isomerization of the central complex (EAB) and product releas e are so rapid that E, EA, and EAB never attain equilibrium, but are kept at near-constant, or steady-state levels (Figure 1-5). This is generally the case when the substrat e concentrations and the values of Km (Michaelis constant) for the substrates greatly exceed the enzyme concentration.43,45,46

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9 E A B B A EA EB EABEPQ P Q Q P EP EQ E A B E EA EABEPQ P Q EQ E (A) (B) A B E F EAFP PQ E (C) FBEQ Figure 1-5. Cleland notation of the three main types of Bi Bi mechanisms. (A) Random, (B) ordered, and (C) Ping-Pong. A and B are the substrates, E is the free enzyme, F is a stable enzyme intermed iate, and P and Q are the products. The two and three letter species are transitory (unstable) complexes.43 Initial Velocity Studies The first step in determining the kinetic mechanism of an enzyme is usually initial velocity studies. Initial velocity refers to the rate of the catalyzed reaction in the absence of products. Generally, the reaction is said to be at initial velocity when less than 10% of the substrates have been used and product fo rmation is still linear. Exceptions from this rule include cases where the equilibrium consta nt for the catalyzed reaction is very small or one of the products is removed from the assay mixture, for example if the product is CO2. The velocity data are usually plotted in double reciprocal plots (Lineweaver-Burk plots) as 1/v vs. 1/[A] or 1/[B] with the other substrate at a fixed concentration. Velocity equations for multisubstrate enzymes have been derived and the microscopic rate constants (k1, k -1, etc.) grouped into kinetic constants (Vmax, Km, etc.) that can be determined by experiments. The ordered Bi Bi mechanism, for example, has ten individual rate constants that can be combined in such a way that the initial forward

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10 velocity in the absence of products can be described by an equation containing only four kinetic constants (Figure 1-6).43,45,46 E A EA B EABEPQ P EQE Q + + + + k1k -1k4k -4kpk -pk2k -2k3k -3 34p -1 max ia t 343p4p4-p1 4-23-2-p3p 34p m m A B 1343p4p4-p2343p4p4-pkkk k V = E ; K = ; kk + kk + kk + kkk kkk + kk + kk kkk K = ; K = kkk + kk + kk + kkkkk + kk + kk + kk maxiammm BBAAB v = VKK+ KA + KB + AB Figure 1-6. Ordered Bi Bi mechanism showing the individua l rate constant s (top), and the equation for the initial forward ve locity in the absence of products (bottom).43,46 The double reciprocal forms of the velo city equation from Figure 1-6 are miamm ABB maxmmax AKKKK 111 = 1 + + 1 + vVKBAVB when [A] is varied and [B] is constant, and mm ia BA maxmaxKK K 111 = 1 + + 1 + vVABVA when [B] is varied and [A] is constant. Therefore, except in the rare case when Kia is very small compared to KmA, the data should give intersecting lines, since both th e slope and intercepts of the 1/v vs. 1/[substrate] plots are affected by the value of the constant substrate concentration. The

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11 lines will intersect above, on, or below the x-axis, depending on the ratio of KmA/Kia (Figure 1-7A).46 The random Bi Bi mechanism gives more complex equations describing nonlinear double reciprocal plots unless one substrate is saturating, and equations for the Ping-Pong mechanism describe parallel lines in th e double reciprocal plots (Figure 1-7B).43,46 1/v 1/[A] 1/v 1/[A] (A) (B)Increasing [B] Increasing [B] Figure 1-7. Lineweaver-Burk plots of initial ve locity data for a bisubstrate enzyme. (A) Intersecting lines indicati ng a random or ordered seque ntial (ternary complex) mechanism. (B) Parallel lines sugg esting Ping-Pong kinetics. The same patterns appear when the other substrate is varied. Product Inhibition Studies Taking the reverse of the catalyzed reaction into consideration, complete velocity equations have been derived and written for A or B as the varied substrate in the presence of P or Q. The effects of P and Q on the slopes and intercepts of the lines in double reciprocal plots can be read from the equatio ns and the type of inhibition predicted. Three main types of inhibitors are recogni zed in enzyme kinetics: (A) Competitive inhibitors only affect the slope, but not the 1/v-axis intercept, (B) uncompetitive inhibitors only affect the intercept, but ha ve no effect on the slope, and (C) mixed-type inhibitors (also known as noncompetitive inhibito rs) affect both the slope and intercept (Figure 1-8).43,46-48

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12 1/v 1/[Substrate] 1/v(A) (B)Increasing [I] Increasing [I]1/v(C)Increasing [I]1/[Substrate] 1/[Substrate] Figure 1-8. Lineweaver-Burk pl ots showing inhibition patterns in enzyme kinetics. (A) Competitive inhibition, (B) uncompetitiv e inhibition, and (C) mixed-type (noncompetitive) inhibition. For product in hibitions I is either P or Q and [A] is varied at constant [B] or vice versa. The complete velocity equation for the ordered Bi Bi mechanism, rearranged to show inhibition by P vs. A, at constant [B] is max mm iamm QQ BB m A miqmiqmip APP Slope factorIntercept factorA v = V KPKP KKK P K1 + 1 + + A1 + 1 + + KBKKBKKK where the indicated slope and intercept factors will be factors in the slope and intercept of the reciprocal equation. Ther efore, at low (unsaturating) [B], the inhibition by P is a mixed-type inhibition since both the slope and in tercept factors are a function of [P]. At very high (saturating) [B], the slope factor approaches unity, but th e intercept factor is still dependent on [P], so the inhibition is uncompetitive.46-48 Rearranged to show inhibition by P vs. B, at constant [A], the equation becomes max m m Q ia A m B iqmip P Intercept factor Slope factorB v = V KP K P K K1 + 1 + + B1 + + AKKAK showing that P is a mixed-type inhibitor at all concentrations of A, since very high (saturating) [A] leaves [P] terms in both slope and intercept factors.46-48

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13 Inhibition by Q vs. A, at c onstant [B] is described by max iamm BB m A iqm A Intercept factor Slope factorA v = V KKK Q K1 + 1 + + A1 + KKBB which shows that Q is a competitive inhibitor at all concentrations of B, since [Q] has an effect on the slope factor at any [B], but has no effect on the intercept factor.46-48 Finally, the inhibition by Q vs. B, at constant [A] is described by max m ia A m B iqiq Slope factorIntercept factorB v = V K QQ K K1 + 1 + + B1 + 1 + AKAK showing that Q is a mixed-type inhibitor at relatively low (unsaturating) concentrations of A with both the slope and intercept factors be ing functions of [Q]. At very high [A] (saturating) there is no inhi bition since the [Q] terms are eliminated from both factors.46-48 Velocity equations for other possible Bi Bi mechanisms show different product inhibition patterns, making it possible to di stinguish between mechanisms solely by product inhibition studies.46-48 Some examples are shown in Chapter 2 (Table 2-5). Research Objective The main objective of this project was to obtain a thorough un derstanding of the mechanism of CoA transfer by formyl-CoA transferase, a key enzyme in oxalate breakdown by Oxalobacter formigenes. A mechanism is proposed based on kinetic and crystallographic data (Figure 3-8). This is th e first detailed mechan istic study of a Family III CoA transferase, and suggests a novel m echanism of CoA transfer likely employed by all members of this class of enzymes.

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14 CHAPTER 2 EXPRESSION, PURIFICATION, AND KINETIC STUDIES OF FORMYL-COA TRANSFERASE The results of kinetic studies and all requirements for the enzyme kinetics are covered in this chapter, incl uding protein expression, protei n purification, synthesis of thioester substrates, and enzyme assay development. Expression and Purification of Re combinant Formyl-CoA Transferase FRC was produced by IPTG (isopropyl--D-thiogalactopyranosid e) induction of Escherichia coli, carrying the FRC gene in a pET-9a vector. This bacterial strain, commonly used for protein expression, has the advantage of being deficient in proteases that might degrade the desired protein, a nd it carries a gene for T7 RNA polymerase, whose transcription is controlled by the IPTG inducible lacUV5 promoter.49,50 Upon induction by IPTG the bacterium starts produ cing T7 RNA polymerase which recognizes a T7 promoter located shortly upstream of th e FRC gene in the pET-9a plasmid. Thus the bacterium starts produci ng FRC after addition of IPTG. The purification of FRC was based on a procedure developed by Kjell Eriksson and Billi Herzer (Fast Trak process development, Amersham Pharmacia Biotech, Inc., currently known as Amersham Biosciences ) for Ixion Biotechnology, Inc (personal communication). The enzyme was purified from crude lysate of harvested cells by two steps of anion exchange, one st ep of affinity chromatography, and a buffer exchange step. Various adjustments were made to the origin al procedure to optimize the yield of active enzyme. The enzyme proved stable enough to do all the FPLC (fast performance liquid

PAGE 26

15 chromatography) work at ambient temperature if it was kept on ice between column runs, and the first three purification steps were performed on the same day. The yield of purified FRC was typically 10-15 mg of highl y pure protein from each liter of culture (Table 2-1 and Figure 2-1). See Appendix A for examples of FPLC chromatograms of the FRC purification. Table 2-1. Purification table of FRC showing typical yield a nd purification level. Some activity measurements were not reliable due to precipitation of protein in the samples prior to assaying, and are omitted from the table. Purification step Total protein (mg) Specific activity (U/mg) Total activity (U) Yield (%U) Purification (fold) Cell lysate 1150 0.51 587 100 DEAE anion exchange 333 BlueFF affinity chromatography and buffer exchange 67 Q Sepharose High Performance (QHP) anion exchange 63 6.11 385 66 12 (A) (B) Figure 2-1. SDS-PAGE gels showing the expr ession level and purific ation of FRC. (A) 1. Cell pellet at time of induction; 2. Ce ll pellet 150 minutes after induction; 3. Molecular weight markers (kDa). (B) 4. Pooled FRC fractions from DEAE column; 5. Empty lane; 6. BlueFF fl owthrough fractions; 7. BlueFF low salt wash; 8. MW markers (same as lane 3A); 9. Pooled FRC fractions after buffer exchange; 10. BlueFF NaOH wash; 11. QHP low salt wash; 12. Fully purified FRC from QHP. Both gels were stained with Coomassie Blue. The selenomethionine derivative of FRC, which was needed to solve the protein crystal structure, was prepared by inhibiting the natural biosynthesis of methionine by the

PAGE 27

16 expression strain grown in minimal medium and adding selenomethionine before IPTG induction.51 The selenomethionine derivative wa s purified as described above for the recombinant wild-type FRC. Mass spect rometry showed full incorporation of selenomethionine. No formyl-CoA transferase activity was detected in a cell lysate of expression strain cells, containing a pET-9a plasmid without the FRC gene, grown under the same conditions as when FRC was expressed. F RC was therefore assumed to be responsible for all the CoA transferase activity is olated from the expression strain. Synthesis of CoA Esters The CoA ester substrate and product were synthesized using a thioester exchange reaction between CoA and an ar omatic thiol ester of the appropriate acid (Figure 2-2). SH HO O O S O H CoASH CoAS H O HO O O O O S O O O CoASH CoAS O O O pH 8 + 45C, 2.5 hrs cat. pyridine (A) (B) pH 8 Figure 2-2. Synthetic schemes for formyl-CoA and oxalyl-CoA. A) Synthesis of formylCoA from CoA and formylated thiophenol B) Synthesis of oxalyl-CoA from thiocresol oxalate and CoA. Formyl-CoA Formyl-CoA was produced by allowing CoA to react with an excess of formylthiophenyl ester as described by Sly and Stadtman.52 The formylthiophenyl ester was made by formylating thiophenol53 using a formylating reagent made from acetic

PAGE 28

17 anhydride and formic acid as de scribed by Stevens and van Es54 (Figure 2-2A). The formylation of CoA was verifi ed by mass spectrometry, and the expected change in retention time on reverse phase C-18 column by HPLC was observed (peaks 3 and 4 in Figure 2-6). Oxalyl-CoA Oxalyl-CoA was synthesized by a sim ilar method, except the precursor was thiocresol oxalate monoester made previous ly by Dr. Jianqiang Wang by the method of Stolle55 (Figure 2-2B). The synthesis was ve rified by mass spectrometry and by HPLC (Figure 2-3). 3 1 (A) 1 (B)(C) 2 3 1 2 4 Figure 2-3. Three HPLC chromatograms showing the hydrolysis of oxalyl-CoA by KOH. (A) Crude oxalyl-CoA reaction mixtur e. (B) Same as (A), but spiked with CoA. (C) Same as (A), but after hydrolysis with KOH. Peak assignments: 1 Oxalyl-CoA; 2 CoA; 3 Thiocresol oxalate monoester; 4 Thiocresol. The chromatograms show absorbance at 260 nm vs. time and all have same or similar scale. Peaks we re assigned by coinjecting standards. The slight discrepancy in retention time s of peak 3 in (A) and (B) is due to instrumentation problems. Purification The excess aromatic thiol esters were removed from the crude reaction mixtures by multiple ether extractions, adjusting the pH of the aqueous phase to 3.0 prior to each

PAGE 29

18 extraction (Figure 2-4). Both CoA esters we re further purified by preparative reverse phase HPLC followed by freeze-drying to mini mize the amount of CoA in their stock solutions. Figure 2-4. Three HPLC chromatograms s howing extraction of thiocresol oxalate monoester from oxalyl-CoA reaction mixtur e. (A) Crude reaction mixture. (B) After four extractions with ether. (C) After eight extractions with ether. Peak assignments: 1 Oxalyl-CoA; 2 Thiocresol oxalate monoester. The chromatograms show absorbance at 260 nm vs. time and all have same or similar scale. Peaks were assigned by coinjecting standards. The slight discrepancy in retention times of peak 2 in (A) and (B) is due to instrumentation problems. Stability Stabilities of the CoA esters under th e assay conditions were determined by measuring the pseudo first-order rate for th e uncatalyzed hydrolysi s of these compounds at pH 6.7 and 30C. The concentrations of the CoA esters were measured by HPLC as described below. The half-lives were cal culated from the pseudo first-order rate constants taken as the slope of the best-fit lin e of the plots in Figur e 2-5, as described by the first-order rate equation: ln[A] = ln[A]0 – kt. This gave an estimate of 150 min for the half-life of formyl-CoA, which is in reas onable agreement with a literature value of 300 min in aqueous solution at r oom temperature and neutral pH.52 The measured half life for oxalyl-CoA was about 10 days, which is al so consistent with previous reports that

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19 solutions of oxalyl-CoA at pH 6.5 are st able for weeks when stored at –15C.10 The large difference in the rate of uncatalyzed hydrol ysis of the two thioesters can likely be attributed to the presence of the negati vely charged carboxylate group in oxalyl-CoA, which will destabilize the tetrahedral adduct formed by nucleophilic attack of water on the thioester carbonyl. (A) 4.3 4.4 4.5 4.6 0204060 t (min)ln[formyl-CoA]t1/2 150 min (B) 4.58 4.59 4.60 0100200300400 t (min)ln[oxalyl-CoA]t1/2 10 days Figure 2-5. Graphs for determining the rate of hydrolysis of the CoA esters. (A) FormylCoA and (B) oxalyl-CoA. Assay Development The only other previous study on FRC used a coupled enzyme assay to determine the rate of formate production by monito ring the formation of NADH from NAD+ by formate dehydrogenase (FDH).11 A more direct approach of determining the rate of oxalyl-CoA formation by HPLC was developed for the studies described here. The FDH coupled assay consistently produced lower rate values than the HPLC-based assay. Since NAD+ was suspected to inhibit FRC due to its st ructural similarity to the CoA esters, the effect of 0.5 and 1.0 mM NAD+ in the HPLC assay was studied. Rapid hydrolysis of formyl-CoA was observed and very little pr oduction of oxalyl-CoA was detected. The hydrolysis of formyl-CoA in the presence of NAD+ may explain the high value of Km for formyl-CoA (3.0 mM) published in the original study of FRC.11 This value is closer to

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20 the Km value of formate for the FDH used (13 mM),56 than the published Km values for CoA esters in two other Family III CoA transferases (3 and 40 M).27,29 (A) 1 2 3 4 (B) (C) Figure 2-6. HPLC chromatogr ams of assay mixture aliquots quenched after 5 min (A), 15 min (B), and 55 min (C). Peak as signments: 1 Oxalate; 2 Oxalyl-CoA; 3 CoA; 4 Formyl-CoA. The chromatograms show absorbance at 260 nm vs. time and all have same or similar scale. Peaks were assigned by injecting standards. The facile separation of CoA and its esters by reverse phase HPLC57 is the basis for the enzymatic assay used in the work descri bed here. The rate of oxalyl-CoA production by the enzyme can be determined from the amount of oxalyl-CoA present in assay mixtures quenched at different timepoints as demonstrated in Figure 2-6. Similar conditions were used as in the previous study, except no other enzyme reaction was coupled to the one being studied. The en zyme, substrates, and inhibitors when appropriate, were added to phospha te buffer at pH 6.7, adding formyl-CoA last to initiate the reaction. Aliquots of the assay mixtur e were quenched with acetic acid at two

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21 different timepoints, typically after 1.0 and 1.5 minutes, st ored on ice, and analyzed by HPLC within 60 minutes. The efficacy of the quench was verified by performing the assay at pH 3-4 instead of pH 6.7. No oxalyl-CoA had formed afte r 28 min, nor after overnight incubation at ambient temperature. Furthermore, the formyl-CoA peak area did not decrease appreciably over the first 28 min, but had dr opped somewhat overnight due to hydrolysis as evidenced by an equal increase in the Co A peak area. After quenching, the pH is between 3 and 4, which is favorable for the stability of CoA esters, thus there was no detectable decrease in oxalyl-CoA or form yl-CoA content in quenched samples stored on ice for up to 60 minutes. Free CoA and CoA esters were quantitated by HPLC using a standard solution of CoA. The assumption was made that the exti nction coefficients of the CoA esters at 260 nm, the detection wavelength, were the same as for CoA. This assumption was successfully validated for form yl-CoA by measuring its conc entration in solution using the hydroxylamine method,58-60 and for oxalyl-CoA by measur ing the concentration of oxalate in an oxalyl-CoA solution before a nd after hydrolysis by base using an oxalate oxidase-based detection kit (Sigma-Aldrich Corp., St. Louis, MO). The rate of the catalyzed reaction incr eased linearly with increasing enzyme concentration (Figure 2-7). Diluting the enzyme stock solution excessively before assaying, however, resulted in lowered speci fic activity (Table 2-2). This most likely resulted from dissociation of the homodimeri c enzyme into its subunits upon dilution, a proposal supported by size exclusion chromatogr aphy studies discussed in Chapter 3. A 20-fold dilution of a 0.90 mg/mL stock enzyme solution retained its specific activity well

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22 over a few days when stored at 4C, while 40fold and 80-fold diluted solutions lost their specific activity rapidly. Typically the 20fold diluted stock solution lost only about 15 % of its transferase activity when stored at 4C for 3 weeks. 0 5 10 15 20 25 0102030405060 [FRC] (g/mL)Rate (M/min ) Figure 2-7. Linear increase of initial rate with increasing enzyme concentration. Table 2-2. Effect of excessi ve dilution of the enzyme stock solution on specific activity. Protein concentration of the original stock was 0.90 mg/mL. The same amount of enzyme was used in all assays. Dilution (fold) Storage time at 0-4C (hours) before assaying Relative specific activity 20X 1 1.0 20X 24 1.0 40X (made from 20X) 1 0.8 40X (made from 1X stock) 24 0.6 80X (made from 40X) 1 0.4 80X (made from 20X) 0 0.9 80X (made from 20X) 1 0.8 80X (made from 20X) 3 0.4 No transferase activity was detected when one of the substrates or the enzyme was not present in the assay mixture, and no activ ity was detected when enzyme denatured by incubation in boiling water for 5 minutes was used. The detection limit of the HPLC assay de pends on the amount of enzyme in the assay mixture, and the length of incubation. Long incubation times are not desirable for initial rate measurements due to the lability of formyl-CoA, but can be used if the goal is

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23 merely to see if there is some measurable activity or not, such as when screening for alternative substrates or checking muta nt enzymes for transferase activity. Equilibrium Constant The equilibrium constant for the FRC-cat alyzed CoA transfer was estimated by measuring the equilibrium concentrations of formyl-CoA and oxalyl-CoA incubated with FRC and known initial amounts of form ate and oxalate. The value of Keq was determined to be 32 3, favoring oxalyl-Co A and formate to formyl-CoA and oxalate. This value is similar to the equilibrium constant determined for the reaction catalyzed by succinyl-CoA:acetoacetate transferase.61 Alternative Substrates Results of the former study identifying succinate and succinyl-CoA as alternative substrates, but not acetyl-CoA,11 were confirmed and a variety of other potential substrates were screened. Somewhat surprisi ngly, the natural substrates, formyl-CoA and oxalate, did not yield the highest rate of CoA transfer. Th e highest specific activities were observed for CoA transfer from succiny l-CoA to formate and from formyl-CoA to succinate. CoA transfer from formyl-CoA to glutarate was also faster than to oxalate, while CoA transfer to maleate was slower Malonyl-CoA, methylmalonyl-CoA, acetylCoA, and propionyl-CoA were not used as CoA donors by FRC with oxalate or formate as acceptors. Propionate, oxamate, and pyruvate were not accepted as substrates (Figure 2-8 and Table 2-3). Acetate did not inhibit the enzyme at c oncentrations of up to 60 mM, while acetylCoA inhibited the rate of CoA transfer by 25% at 100 M and 45% at 200 M concentration in the presence of 35 M fo rmyl-CoA and 100 mM oxalate. There was no indication of an irreversible inhibition by acetyl-CoA, since the enzyme was preincubated

PAGE 35

24 with the inhibitor before adding formyl-CoA. Acetyl-CoA therefore appears to bind to FRC but the thioester is not lysed by the enzyme. This is not surprising since the homologous protein from E. coli has been crystalli zed with bound acetyl-CoA.41 The remaining alternative substrates were not as sayed for inhibition, but it seems likely that any CoA ester will inhibit the enzyme by competing for the CoA binding site with the natural substrate even if the thioester moiety cannot be used as substrate. Table 2-3. Summary of altern ative substrates screening. CoA ester concentrations were 80-200 M and free acid concentrations were 62.5-125 mM. CoA Donor Acceptor (free acid) Approx. specific activity* (mol/minmg) Formyl-CoA Oxalate 5.5 (forward reaction) Oxalyl-CoA Formate 0.7 (reverse reaction) Succinyl-CoA Oxalate 4.5 Succinyl-CoA Formate 40 Formyl-CoA Succinate 50 Formyl-CoA Glutarate 15 Formyl-CoA Maleate 2 Formyl-CoA Oxamate 0 Formyl-CoA Pyruvate 0 Formyl-CoA Acetate 0 Oxalyl-CoA Acetate 0 Malonyl-CoA Formate 0 Malonyl-CoA Oxalate 0 Methylmalonyl-CoA Formate 0 Methylmalonyl-CoA Oxalate 0 Acetyl-CoA Formate 0 Acetyl-CoA Oxalate 0 Propionyl-CoA Formate 0 Propionyl-CoA Oxalate 0 *The limit of detection was approximately 0.001 mol/min*mg The overall selectivity rule for CoA tr ansfer by FRC based on the observations above appears to be a requirement for a ne gative charge on the th ioester end of the incoming CoA ester substrate, and the free acid substrate needs to be a dianion. The only exceptions to this rule are formate/formyl-CoA, which are accepted, and (methyl)malonyl-CoA, which are not accepted as substrates. The exceptions can be explained by the small size of formate, which allows it to position itself correctly in the

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25 active site, and by the lack of flexibility of (methyl)malonyl-CoA, making it unable to attain a favorable conformation in the active site In this regard the large difference in the transfer rates of CoA to succi nate and maleate also indicate s a requirement of flexibility in the CoA acceptor molecule allowing it to make favorable interactions in the active site. The lack of CoA transfer from propionyl-CoA and acetyl-CoA are consistent with these substrates not being able to attain a favorab le conformation in the active site, presumably due to their hydrophobic methyl and ethyl groups. Finally, th e absence of CoA transfer to acetate, oxamate, and pyruvate are all i ndications that without a second negative charge these molecules are not able to make favorable interactions in the active site. H O O O O O O O O O O O O O O R O O O O O O O O C H3 O O O O H2N O O O H3C O O O C H3 Formate Oxalate Succinate Maleate Glutarate Acetate Oxamate Malonate (R = H) Methylmalonate (R = CH3) Pyruvate (A) (B) Propionate Figure 2-8. Structures of free acids and este r parts of CoA esters used for alternative substrate screening (see Table 2-3). (A ) Accepted by FRC as substrates. (B) Not accepted by FRC as substrates. Kinetics As discussed in Chapter 1, kinetic studies are a powerful tool to explore possible mechanisms of enzyme catalysis. In the case of bisubstrate enzymes yielding two products, such as FRC, the pattern of lines in a Lineweaver-Burk plot (1/v vs. 1/[S]) of initial velocity data are a good indication of what type of mechanism the enzyme uses. Intersecting lines are indicativ e of a sequential (ternary complex) mechanism, while

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26 parallel lines suggest Ping-P ong kinetics. In the case of ternary complex mechanisms, random and ordered ones can be distinguish ed by product inhibition patterns, and the order of substrate binding and product release can be deduced in the case of ordered. Initial Rates Initial rate measurements in the absence of products were designed so the same assays would provide data that could be pl otted as 1/v versus 1/[formyl-CoA] and 1/v versus 1/[oxalate] (Figures 2-9 and 2-10). The results were clearly indicative of a sequential (ternary complex) mechanism and ruled out Ping-Pong kinetics. The kinetic constants kcat, Vmax, Km(formyl-CoA), Km(oxalate), and Kia (dissociation constant for the EA complex) were calculated from the slope and intercept replots (Figures 2-9 and 2-10 inserts), and are summarized in Table 2-4. The Km of formyl-CoA in this study is about 400-fold lower than that reported for the native FRC, while the Km of oxalate is comparable (Table 1-1). Descriptions of how to calculate the kinetic constants from bestfit lines of double reciprocal plots and th eir replots are accessible in Segel’s book on enzyme kinetics.46 Error values were calculated by standard error propagation rules from the original standard errors in slopes and inte rcepts of the best-fit straight lines fitted by using KaleidaGraph (v. 3.5, Synergy Software). Table 2-4. Kinetic constants of FRC calculate d from initial rates data (Figures 2-9 and 210). One unit (U) is defined as one mi cromole of CoA transferred per minute. Constant Value kcat 4.3 0.1 s-1 Vmax 5.5 0.2 U/mg Km(formyl-CoA) 8.0 0.3 M Km(oxalate) 3.9 0.3 mM Kia 16 2 M

PAGE 38

27 -0.1 0.2 0.4 0.6 0.8 -0.25-0.15-0.050.050.15 1/[oxalate] (mM-1)1/v (min mg mol-1) 0.1 0.2 0.3 0.4 0.00.1 1/[f-CoA] 0.5 1.0 1.5 2.0 2.5 0.00.1 1/[f-CoA] Figure 2-9. Lineweaver-Bur k plot of initial rates at three fixed formyl-CoA concentrations ( 7 M; 14 M; 65 M), each at four oxalate concentrations (5 – 75 mM). Slope and in tercept replots are shown as inserts. Each data point represents an average of two rate measurements. -0.1 0.2 0.4 0.6 0.8 -0.15-0.050.050.15 1/[formyl-CoA] (M-1)1/v (min mg mol-1) 0.2 0.3 0.4 0.00.10.2 1/[oxalate] 1.5 2.0 2.5 3.0 3.5 0.00.10.2 1/[oxalate] Figure 2-10. Lineweaver-Burk plot of initial rates at four fixed oxalate concentrations ( 5 mM; 10 mM; 25 mM; 75 mM), each at three different oxalate concentrations (7 – 65 M). Slope and in tercept replots are shown as inserts. Each data point represents an average of two rate measurements.

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28 Product Inhibition The appropriate product inhibitions by fo rmate and oxalyl-CoA that would support or rule out the proposed mechanism (Figur e 3-8) were studied. The results are summarized in Tables 2-5 and 2-6 and discussed below. Table 2-5. Product inhibition patterns obser ved for various mechanisms of bisubstrate enzymes. The inhibitions observed in this study are shown in bold italic letters. Substrates and products ar e A = formyl-CoA, B = oxalate, P = formate, and Q = oxalyl-CoA. The types of inhibitions are C = competitive, MT = mixed-type (noncompetitive), UC = uncompetitive, and = no inhibition. Table reproduced fr om Segels book on enzyme kinetics.46 Varied substrate A B Mechanism Inhibitor Unsaturated with B Saturated with B Unsaturated with A Saturated with A P MT UC MT MT Steady-state Ordered Bi Bi Q C C MT P MT MT MT MT Steady-state Random Bi Bi Q MT MT MT MT P MT C C Steady-state Ping Pong Q C C MT P C C Rapid equilibrium Random Bi Bi Q C C Table 2-6. Inhibition consta nts for formate and oxalyl-CoA.* Inhibition constant Value Slope (Kis) and intercept (Kii) composition of Ki Ki(formate vs. formyl-CoA) 130 20 mM N/A (pure uncompetitive) Kis = 17 1 mM Ki(formate vs. oxalate) 350 30 mM Kii = 380 40 mM Kis = 150 50 M Ki(oxalyl-CoA vs. oxalate) 21 7 M Kii = 280 90 M *Inhibition constants were calcu lated as described by Segel46 and error values were calculated as described above. Product inhibition by formate was measur ed under three different conditions (Figures 2-11, 2-12, 2-13). Only one se t of conditions was practical for measuring product inhibition by oxalyl-CoA (Figure 2-14), that is varying oxalate concentration at fixed, unsaturating formyl-CoA concentra tion. Making a stock solution with high

PAGE 40

29 enough concentration of formyl-CoA to achie ve saturation in the assay mixture is difficult, and the experiment would waste large amounts of the relatively expensive substrate without giving much information a bout the mechanism (Table 2-5). OxalylCoA could not be studied as a product inhibito r when varying formyl-CoA concentrations since the initial rate of formation of oxalyl -CoA is being measured, only a relatively large increase in its concentration can be detected when the assay mixture already contains it. This is not possible at low formyl-CoA con centrations because it would require using 20100% of the substrate, and the reaction woul d therefore not be at initial velocity. Inhibition by formate Formate caused mixed-type inhibition when the concentration of formyl-CoA was varied at a fixed unsaturati ng oxalate concentration of 350 mM (Figure 2-11), but the inhibition became uncompetitiv e with oxalate saturating (1.25 M) (Figure 2-12). 0.0 0.2 0.4 0.6 0.8 -0.050.000.050.100.150.20 1/[formyl CoA] (M-1)1/v (min mg mol-1) Figure 2-11. Lineweaver-Burk plot showi ng product inhibition by formate vs. formylCoA at fixed unsaturating oxalate concentration. Formate concentrations were 0 mM, 75 mM, and 150 mM. Formyl-CoA concentrations were 5.5 – 110 M. The initial concentration of oxalate was 350 mM in all assays. Each data point represents an average of two rate measurements.

PAGE 41

30 0.0 0.1 0.2 0.3 0.4 0.5 -0.10-0.050.000.050.100.150.20 1/[formyl CoA] (M-1)1/v (min mg mol-1) 0.15 0.20 010203040 [formate] (mM) Figure 2-12. Lineweaver-Burk plot showi ng product inhibition by formate vs. formylCoA at fixed saturating oxalate concentra tion. Formate concentrations were 0 mM, 20 mM, and 40 mM. Formyl-CoA concentrations were 5.5 – 110 M. The initial concentration of oxalate was 1.25 M in all assays. Intercept replot is shown as insert. Ea ch data point represents an average of two rate measurements. 0.0 0.4 0.8 1.2 -0.10.00.10.20.30.4 1/[oxalate] (mM-1)1/v (min mg mol-1) 0.18 0.22 050100 [formate] 0 5 10 050100 [formate] Figure 2-13. Lineweaver-Burk plot showing product inhibition by formate vs. oxalate at fixed unsaturating formyl-CoA concentra tion. Formate concentrations were 0 mM, 50 mM, and 100 mM. Oxalate concentrations were 2.5 – 230 mM. The initial concentration of fo rmyl-CoA was 105 M in all assays. Slope and intercept replots are shown as in serts. Each data point represents an average of two rate measurements.

PAGE 42

31 Formate also caused mixed-type inhibition when the concentration of oxalate was varied in the presence of fixed unsaturati ng formyl-CoA concentration (Figure 2-13). The inhibition was close to being pure comp etitive, since the slope effect was much greater than the intercept effect (Table 2-6). Inhibition by oxalyl-CoA Oxalyl-CoA functioned as a mixed-type inhi bitor when the concentration of oxalate was varied in the presence of fixed unsatura ting formyl-CoA concentr ation (Figure 2-14). Relatively high concentrations of oxalyl-CoA were needed to see any inhibition, which indicates the inhibition will disappear at sa turating formyl-CoA concentration, although this is hard to confirm since very high formyl-CoA concentration is likely needed. -0.2 0.2 0.6 -0.3-0.2-0.10.00.10.20.30.4 1/[oxalate] (mM-1)1/v (min mg mol-1) 0.15 0.25 0.35 0100200 [oxalyl-CoA] Figure 2-14. Lineweaver-Burk plot show ing product inhibition by oxalyl-CoA vs. oxalate at fixed unsaturating formyl -CoA concentration. Oxalyl-CoA concentrations were 0 M, 90 M, and 180 M. Oxalate concentrations were 2.5 – 75 mM. The initial concen tration of formyl-CoA was 100 M in all assays. Intercept replot is shown as insert. Each data point represents an average of two rate measurements. The kinetic measurements of FRC detail ed above strongly support an ordered ternary complex mechanism where formyl-CoA binds first, followed by oxalate, with

PAGE 43

32 formate then being released before oxalyl-C oA. Other possible kinetic mechanisms are effectively ruled out by the results above (Tab le 2-5). The mechanism is discussed in more detail in Chapter 3. Inhibition by Coenzyme A CoA was of interest as a potential inhibito r, since inhibition by CoA would indicate binding of it to the active site and therefore a possibility of seeing it bound in the crystal structure of FRC incubated with CoA. This would be an example of dead-end inhibition assuming CoA binds to the free enzyme in th e same way formyl-CoA does, but yielding an enzyme-inhibitor complex that is unable to do catalysis. A mixed-type inhibition pattern was observed when CoA was used as an inhibitor against oxalate at fixed unsaturating formyl-CoA concentration is show n in Figure 2-15. This is the expected pattern when the inhibitor binds only to the free enzyme, and further supports an ordered Bi Bi mechanism for FRC.62 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 -1.20-0.70-0.200.30 1/[oxalate] (mM-1)1/v (min mg mol-1) Figure 2-15. Lineweaver-Burk plot showi ng inhibition by CoA vs. oxalate at fixed unsaturating formyl-CoA concentration. CoA concentrations were 35 M, 75 M, and 115 M. Oxalate concentrations were 2.5 – 50 mM. The initial concentration of formyl-CoA was 75 M in all assays. Each data point represents an average of two rate measurements.

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33 CHAPTER 3 STRUCTURE AND MECHANISM OF FORMYL-COA TRANSFERASE At the start of this research project, Fa mily III CoA transferases had not been recognized as a new class of enzymes and protei n structures were not available. Crystal structures of FRC as an apoenzyme and in complex with CoASH became available as a result of collaboration with a research group at the Karo linska Institute in Stockholm, Sweden.42,63 Further crystallization experiment s yielded a crystal structure of an acylenzyme intermediate formed when FRC was incubated with oxalyl-CoA.64 These structures and those of three mutant enzymes* are discussed below and how they, combined with kinetic data from Chapter 2, lend strong support for a novel mechanism of CoA transfer. Structure The crystal structure of FRC reveals a homodimer with a unique assembly of the subunits. Each monomer consists of a large and a small domain where residues from both the Nand C-termini of the subunit ar e part of the large domain. The linkers between the domains give the subunit an oval shape with a 13 by 22 hole in the middle. In the homodimer the second monomer is threaded through th e hole in the first one and vice versa like two li nks in a chain (Figure 3-1). Structure factors and coordinates have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) under accession codes 1P5H (FRC), 1P5R (FRC/CoA comp lex), 1VGQ (D169A mutant), 1VGR (D169E mutant), 1T3Z (D169S mutant) and 1T4C (FRC/oxalyl-CoA complex).

PAGE 45

34 The interlocked dimer that is observed in the crystal structure of FRC is inconsistent with previous claims that th is enzyme exists as a monomer in solution.11 The oligomeric state of recombinant wild-t ype FRC was therefore examined using size exclusion chromatography. (A) (B) Figure 3-1. Monomer and homodimer structures of recombinant formyl-CoA transferase. (A) One monomer of FRC revealing a la rge hole in the oval structure. The protein is shown as molecu lar ribbons with the N-termi nus in blue and gradual color change through cyan, green, and yello w to the C-terminus shown in red. (B) The structure of the interlocke d FRC homodimer with bound CoA. For clarity, the protein monomers are colo red red and green and represented by molecular ribbons. The bound CoA molecules are shown as space-filling models, which are colored using the following scheme: H white; C grey; N blue; O red; S yellow; P purple. When 7 g of FRC were injected on the size exclusion column a broad peak with a retention time corresponding to a molecular we ight of 53.8 kDa resulted. Larger amounts (30 g) of FRC, however, yielded a sharp p eak matching a mass of 81.0 kDa (Figure 33B). The smaller mass (53.8 kDa) is reasona bly consistent with the mass of a monomer (47.2 kDa) with the deviation between meas ured and theoretical mass arising from the open structure of the monomer, and the broadne ss of the peak probably a result of various

PAGE 46

35 states of unfolding (Figure 3-1A). The larger mass, similarly, is consistent with the enzyme being a homodimer with the deviation between measured (81.0 kDa) and theoretical mass (94.4 kDa) aris ing from the tightly interloc ked dimer structure (Figure 31B). This is consistent with the obser vation that excessive dilution of FRC causes decreased specific activity of the enzyme (see Chapter 2), and most likely means the homodimer is disassociating on the size exclus ion column when small amounts of sample are injected. 4.0 4.5 5.0 5.5 00.10.20.30.40.5KDlog(MW)Homodimer (81.0 kDa) Monomer (53.8 kDa) Figure 3-2. Size exclusion chromatography data used to calculate the molecular mass of FRC. Retention coefficients (KD) of molecular weight standards are shown by filled circles. Dotted lines show the observed KD for samples of FRC and the value of log10(MW) calculated from the equation of the best-fit line through the filled circles. Open circles indicate the placement of FRC samples on the calibration line. KD is calculated as (elution volume – void volume)/(column volume – void volume). (A) (B) Figure 3-3. Size exclusion HPLC chroma tograms of FRC. (A) Broad peak from smaller amounts of FRC corresponding to 53.8 kDa. (B) Sharp peak from larger amounts of FRC corresponding to 81.0 kDa.

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36 Active Site There are two equivalent CoA binding sites in the FRC homodimer, located at the interface between the large domain of one subunit and the small domain of the other subunit (Figure 3-1B). The adenine part of CoA is wedged into a thin cleft and buried from solvent while the ribose, ribose phospha te, and pyrophosphate are solvent-exposed. The pantetheine chain of CoA is buried in a cleft formed mainly by the large domain. The small domain participates with a loop composed of residues 258-261 closing off the cleft where the sulfhydryl group on the pant etheine arm of CoA is bound. In the apoenzyme structure, this loop adopts an open conformation in one subunit, but has a closed conformation in the other. In the F RC-CoA complex, this l oop is in the closed conformation in both monomers, leaving insuffi cient space for an oxalate molecule to bind in the active site. There is however a suit able binding site for oxalate in the vicinity of the sulfhydryl group when the loop is in the open conformation (Figure 3-7). Figure 3-4. Stereo picture of Coenzyme A in its binding s ite. Amino acids involved in hydrogen bonds with CoA are colored according to atom type (C atoms cyan for FRC and silver for CoA).

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37 FRC M T ------K PL D G I N VL D F TH V Q A G PA CTQ MM G FL G A N VI K I E RR GSG 42 YfdW M S ------T PL QG I K VL D F TG V Q SG P SCTQ MLAWF G A D VI K I E R P G V G 42 BbsF MP NS I E --R AL E G IVV C D F S WV G A G PIA TS VLA QCG A D VI K I E S V KR P 46 FldA M E NN T --N MF SG V K VI E LA N FI AAPAA G R FFA D GG A E VI K I E S P A G 44 CaiB M D H LPMP K F G PLA G L R VV F SG I E IA G PFA GQ MFA E W G A E VIWI E N VAWA 49 : : :: : .* . : ** : ** ** FRC D M T R G W L Q D K P N V D S L Y F T MF NCN KR S I E L D M K T P E G K E LL E Q M 86 YfdW D V T R HQ L R D I P D I D AL Y F T ML NSN KR S I E L NT K T A E G K E VM E K L 86 BbsF D T L RR G E PF K D G I GT G L D R SGY FAA R N A N KR D IAL D M SH P R A R E VAV R L 95 FldA D PL R YT A P S E G R PL SQ EE NTTY D L E N A N KK AIVL N L K S E K G KK IL H E M 92 CaiB D T I R ---V Q ---P --N --Y P Q L S RR N L H AL S L N IF K DE G R E AFL K L 85 : : : : : .. :: : FRC I KK A D VMV E N F G P G AL D R M G F T W E Y I Q E L N P R VILA -S V K GY A E GH A 132 YfdW I R E A D ILV E N F H P G AI D H M G F T W E H I Q E I N P R LIF G -S I K G F DE CS P 132 BbsF I E K S D IVI NN F R V GQ M E K W K L G W ED V Q K I N P R AI Y V T M S M QG I D G -P 141 FldA LA E A D ILL TN W R T K ALV K QG L D Y E T L K E K Y P K LVFA -Q I TGYG E K G P 138 CaiB M E TT D IFI E A S K G PAFA RR G I T DE VLW QHN P K LVIA -H L SG F GQYGT EE 133 : : : : : : : : : : : : : FRC N E H L K V Y E N V A QCSGG AAA TTG FW D G -PP T V SG AAL G D S NSG M H LM 177 YfdW Y V N V K A Y E N V A Q AA GG AA STTG FW D G -PPLV S AAAL G D S NTG M H LL 177 BbsF HS R Y M GYG V N L N AL C -G L T A R A G F P GQ APF GTGTNYT D H VMVP THT L 187 FldA D K D LP G F D YT AFFA R GG V SGT L Y E K GT VPP N VV P G L G D H Q A G MFLA 184 CaiB YTN LPA Y NT I A Q AF SGY LI QNG D V D --Q PMPAFP YT A D Y F SG L T A T 177 : * * FRC I G ILAAL E M R H K TG R GQ K VAVAM Q D AVL N LV R I K L R D QQ R L E R TG ILA E 226 YfdW I G LLAALL H R E K TG R GQ R V T M S M Q D AVL N L C R V K L R D QQ R L D K L G Y L EE 226 BbsF F G IMAALL E R E A TG R GQT V S L SQ L E S AI -C M T P S APMAFAA NG E AL G 233 FldA A G MA G AL Y K A K TTGQG D K V T V S LM HS AM YG L G IMI Q AA Q Y K D HG -LV 230 CaiB T AALAAL H K A R E TG K G E S I D IAM Y E VML --R M GQY FMM D Y F NGG E M C 222 .** ** : : : :: : : : FRC Y P Q A Q P N FAF D R D GN PL S F D N I TS VP R GGN A GGGGQ P G WML K C K G W E T 274 YfdW Y P Q Y P N -----G --T F G D -AVP R GGN A GGGGQ P G WIL K C K G W E T 262 BbsF P Q ---------G ---YG D P E AAP HG V YT ---T L G Y -R -K WI 257 FldA Y PI ---------N ----R N -E T P N PFI ----V S Y -K S K -D 250 CaiB P R M S K -----G ----K D ---P Y -Y A G ---CG L Y -K C A D GY I 246 : : : FRC D A D SY V Y F T IAA N MWP Q I -C D MI D K P E W K DD PA Y NT F E G R V D K LM D I 320 YfdW D P N A Y I Y F T I Q E QN W E NT -C K AI G K P E WI T D PA Y ST A H A R Q P H IF D I 308 BbsF AIA V F D D A Q WA T L -RR VM GN PPWA EDE R F A T I E M RRR H AA E L 298 FldA D Y FV Q V C MPP Y D VF Y D R F -M T AL G R ED LV G DE R Y N K I E N L K D G R A K E V 297 CaiB VM E LV G I TQ I EE C F K D I G LA H LL GT P E IP E GTQ LI H R I E C P YG PLV E 293 : : : : . : FRC F S FI E T K F A D K D K F E V T E WAA QYG IP CG PVM S M K E LA H D P S L Q K V GT 367 YfdW FA E I E K YT V T I D K H E AVA Y L TQ F D IP C APVL S M K E I S L D P S L R QSGS 355 BbsF DE R I E G W -T A TQYG D WLM E ALL K A G VAA G E V R D A R E AI EDE H L RRR G F 345 FldA YS II E QQ M V T K T K D E W D N IF R D A D IPFAIA QT W ED LL EDE Q AWA N D Y 344 CaiB E K L D AW LAA HT IA E V K E R FA E L N IA C A K VL T VP E L E SN P QY VA R E S 339 :: : . : . : : FRC VV E VV DE I R GN H L T V G AP F K -F SG F Q P E I T R APLL G E HT DE VL K 410 YfdW VV E V E Q PL R G K Y L T V GC P M K -F S AF T P D I K AAPLL G E HT AAVL Q 398 BbsF WA Y L D H P E V G V T L YN R AP IV -F S R -T PV E M K S AAP S I GQHT R E VL G 389 FldA L Y K M K Y P TGN E R ALV R LP VF -F K E A G LP E YN QS P Q IA E NT V E VL K 388 CaiB I TQ W QT M D G R --TC K G P N IMP K F K N -N P GQ IW R G MP SHG M D T AAIL K 383 * *. : .* : FRC E L G L DD A K I K E L H A K Q VV ----428 YfdW E L GYS DDE IAAM K QNH AI ----416 BbsF G ML GYSHG E I ED L AA QQ VLV ---409 FldA E M GYT E Q E I EE L E K D K D IMV RK E K 412 CaiB N I GYS E N D I Q E L V S K G LA K V ED 405 : .* : : Figure 3-5. CLUSTAL W (1.82) multiple sequence alignment of Family III CoA transferases. FRC (formyl-CoA transferase from O. formigenes), YfdW (the FRC homolog from E. coli), BbsF (succinyl-CoA: (R)-benzylsuccinate CoA transferase from Thauera aromatica), FldA ((E)-cinnamoyl-CoA: (R)phenyllactate CoA transferase from Clostridium sporogenes), CaiB (butyrobetainyl-CoA: (R)-carnitine CoA transferase from E. coli).

PAGE 49

38 The residues involved in bindi ng of CoA are identified in Figure 3-4. Asp169 and Gln17 are closest to the sulfhydr yl end of CoA and Asp169 is in a position where it could attack the carbonyl group of a bound CoA ester. Multiple sequence al ignment of Family III CoA transferases is shown in Figure 3-5. YfdW from E. coli is likely a formyl-CoA transferase since it shares 60% sequence identity with FRC. FRC shared considerably lower sequence identy with the other transf erases, or 26%, 23%, and 20% with BbsF, FldA, and CaiB respectively (see abbrev iations in Figure 3-5 legend). Asp169 was identified as a residue potentially playing a critical role in catalysis by FRC due to its position in the active site and because it is c onserved in Family III CoA transferases. The only other residue close to the sulfhydryl group of bound CoA that is fully conserved is Pro20, which is likely needed for structural purposes creating a critical loop in the structure. Tyr59 and Tyr139, which are also cl ose to the active site, are conserved in four of five sequences shown in Figure 3-5. Ty r59 has been suggested to participate in stabilizing the oxyanion tetrahedral intermediates that may be formed in the transfer reaction.42 Tyr139 makes hydrophobic contacts with the dimethyl group of the pantetheine chain of bound CoA. Asn96, which c ontacts CoA, is also conserved in four of the five sequences. The relatively low conservation of residues in the active site of Family III CoA transferases reflects the broad ra nge of substrates that these enzymes use. The location of the active site, in a cleft be tween a large domain and a sma ll domain with the sulfhydryl end of CoA pointing towards the small domain (Figure 3-1B), suggests that the difference in substrate selectivity between the member s of Family III CoA transferases may be linked to structural differences in the small subunits. This would allow the same overall

PAGE 50

39 protein fold for all members, while creating flexibility in active site structure. As described above, the small subunit of FRC is part of the active site st ructure via a flexible tetraglycine loop consisting of residues 258-261. This moiety is also seen in YfdW, but not in the other Family III CoA transferases in Figure 3-5, where there is a gap in the sequence alignment with FRC. This sugge sts a large difference in the active site structures, which would explain the differe nce in natural substrates between these enzymes. Point Mutation Studies Point mutations of FRC were done wher e Asp169 was replaced with alanine, glutamate, and serine to give D169A, D169E and D169S mutants, respectively. The mutations were achieved by using mutage nic DNA primers in a polymerase chain reaction (PCR) with the wild-type FRC in a p ET-9a plasmid isolated from the bacterial expression strain as template. The mutant proteins exhibited the same chromatographic properties as the wild-type FRC during their purifications. The mutations were confirmed by DNA sequencing of the mutant plasmids and, later, by X-ray crystallography (Figure 3-6). The catalytic activity of the mutated prot eins was then assayed using the HPLCbased assay described in Chapter 2. The sens itivity of the assay allowed up to a 30,000fold reduction of CoA transfer rate, relative to wild-type FRC, to be detected. As expected, the mutations dramatically decrease d the transferase activity of the protein. Surprisingly though, while both the D169E and D169S FRC mutants exhibited no activity above the detection limit, the specific activity of the D169A mutant was decreased only 1,300-fold compared to the wi ld-type FRC. No increase in the rate of formyl-CoA hydrolysis was detected in these assays, indicating the enzyme had not been

PAGE 51

40 changed to a CoA ester hydrolase by the mutations. Finally, to ensure the loss of CoAtransferase activity was not due to incorrect folding or quaternary structure of the FRC mutants, X-ray crystal structures of all th ree mutants with bound CoA were obtained. These studies showed that the mutants were correctly folded and formed interlocked dimers like wild-type FRC. In addition, none of the complexes showed any significant difference in structure from that observed fo r the wild-type FRC/CoA structure (Figure 36). Figure 3-6. Stereo images of active site st ructures of FRC mutant s complexed with CoA. Superposition of the active sites of th e CoA complexes of wild-type FRC and (A) D169A, (B) D169S, (C) D169E. The carbon atoms in the wild-type FRC/CoA complex are drawn in cy an, and the mutants in purple. In the complexes involving the D169A a nd D169S FRC mutants (Figure 3-6A/B), the sulfhydryl group of CoA appeared to be oxidized, resulting in hydrogen-bonding with Glu140 rather than Gln17 and Ala18 as obser ved in the wild-type FRC/CoA complex. (A) ( C ) (B)

PAGE 52

41 The reason for this observation only in these two complexes is probably because removal of the Asp169 carboxyl group leaves the th iol group exposed to oxidation. The significant loss of transferase act ivity in these two mutants most likely arises from their inability to form the key anhydride intermed iates (Figure 3-8). The most surprising observation was the detection of any tran sferase activity by D169A, which lacks the active site carboxylate that is apparently critical for normal ac tivity. Since control experiments ruled out contamination by other CoA transferases, the simplest explanation for this observation is that the transfer reaction proceeds by a different mechanism, similar to the one observed for Family II CoA transferases (Figure 1-3B). Thus, oxalate directly attacks formyl-CoA in a ternary co mplex to give an oxalyl-formyl anhydride, which then reacts with bound CoA to yield oxalylCoA and formate. No rate increase of formyl-CoA hydrolysis was observed when this substrate was incuba ted with oxalate and D169A, which is consistent with the absence of water molecules in the active site of D169A observed in its crystal structure. Mo reover, without wild-typ e FRC or the mutant, no oxalyl-CoA formation was detected when formyl-CoA and oxalate were incubated under the standard assay conditions. This fi nding is consistent with previous model studies on the rate of reaction of carboxylic acids with thioesters.65 The conformation of CoA in the active s ite of the D169E mutant was the most similar to that observed for the wild-type FRC/CoA complex. However, since the glutamate side chain is bulkier than that of aspartate the pantotheine group was displaced slightly (Figure 3-6C), and cau sed some uncertainty in positioning of the thiol. The lack of activity in the D169E mutant is probabl y associated with problems in positioning the

PAGE 53

42 formyl-CoA correctly to permit anhydride form ation by reaction of the thioester with the carboxylate moiety (Figure 3-8). Mechanism The mechanism for Family III CoA transf erases proposed by Heider was based on the mechanism of Family II enzymes (Figure 1-3B).25 The enzyme was proposed to catalyze direct nucleophilic a ttack of oxalate on formyl-CoA to yield a mixed oxalylformyl anhydride intermediate and free CoA. Addition of CoA at the other end of the mixed anhydride would then complete th e acyl transfer. Notably, no covalent intermediates would be formed by reaction of th e substrates with active site residues in such a mechanism, the function of the catal yst being primarily to bring the reactants together in the correct orientation. The cr ystal structure of wild-type FRC is, however, not supportive of this mechanism since the pos ition of the side chain of Asp169 appears to prevent a direct attack of oxa late on formyl-CoA (Figure 3-7).42 Figure 3-7. Stereo picture of the end of th e pantetheine chain of CoA and amino acids in the surrounding active site. Superim posed to loop 258-261 in the closed conformation is the same loop in the open conformation as seen in the apoenzyme structure (grey). The two conformations correspond to different rotamer conformations of Trp48. The cavity formed when the loop is in the open conformation is shown as a lig ht green cloud. A model of bound oxalate, in magenta, is include d but its orientation is unknown.

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43 Asp O O R1 SCoA O Asp O O R1 O O R1 O R2 O O R1 O O R2 O O Asp O O R2 O Asp O O R2 SCoA O R2 SCoA O R1 SCoA O -SCoA-SCoA Figure 3-8. Proposed mechanism of formyl-CoA transferase from Oxalobacter formigenes. The box highlights the anhydride intermediate observed by X-ray crystallography (Figure 3-9). The natu ral substrates are R1 = H and R2 = COO. Oxygen atoms from the incoming free acid are shown in bold font. A new hypothesis for the catalytic mechan ism of FRC, which includes the direct involvement of Asp169, has conseque ntly been proposed (Figure 3-8).42,64 Formyl-CoA binds to the active site in the initial step and the substrate th ioester reacts with the Asp169 side chain to form a covalent formyl-Asp169 anhydride intermediate and CoA. Oxalate, entering through the cavity formed when the loop of residues 258261 is in the open conformation (Figure 3-7), reacts to gene rate a new enzyme anhydride intermediate (oxalyl-Asp169 anhydride) and formate. S ubsequent attack of bound CoA on the mixed anhydride then yields oxalyl-C oA and regenerates the car boxylate moiety of Asp169. Since free formate is not produced until af ter oxalate is bound, kinetic plots cannot assume the form observed for classical Ping-Po ng mechanisms like in the case of Family I CoA transferases.

PAGE 55

44 The proposed mechanism of Family III enzymes predicts incorporation of 18O into the main catalytic residue (Figure 3-8) as for Family I enzymes (Figure 1-3A) if an 18Olabeled CoA acceptor would be used. This has not yet been verified for any Family III CoA transferase. An important difference between the proposed mechanism of Family III CoA transferases and the Family I CoA transferase mechanism is the absence of covalent enzyme-CoA intermediates in the former. Family I CoA transferases are completely inactivated when incubated with hydroxylamine in the presence of one of their CoA ester substrates. The inactivati on is a result of the reaction of hydroxamic acids, formed from the hydroxylamine and the th ioester substrate, with the CoA ester of the main catalytic residue (a glutamic acid) (Figure 1-3A).34 Studies on two Family III enzymes support the absence of covalent enzyme-CoA intermediates in the mechanism of Family III CoA transferases. Neither BbsF (succinyl-CoA: (R)-benzylsuccinate CoA transferase) nor FldA ((E)-cinnamoyl-CoA: (R)-phenyllactate CoA transferase) are inactivated by hydroxylamine in tests with and without the CoA ester substrate.27,29 NaBH4 has also been shown to inactivate Family I enzymes by reacting with the covalent enzyme-CoA intermediate reducing the glutamic acid residue to an alcohol.33,35 Family III CoA transferases are at least partially inactivated by NaBH4, although inactivation requires higher concentration of NaBH4 than for Family I enzymes. The activity of BbsF decreased by 75% after in cubation with 0.25 mM benzylsuccinyl-CoA and 1 mM NaBH4 at pH 7.5 for 10 minutes27 and the activity of FldA decreased by 50% after incubation with 49 M cinnamoyl-CoA and 10 mM NaBH4 at pH 7 for 15 minutes.29

PAGE 56

45 The simplest interpretation of these obser vations is that there is no covalent enzyme-CoA intermediate present during cata lytic turnover of Family III CoA transferase enzymes, which would explain them not being inactivated by hydroxylamine. Furthermore, the relatively slow inactivation by NaBH4 is then due to the reduction of a covalent enzyme-substrate anhydride intermedia te that is less reactive and/or not as accessible as the covalent enzyme-CoA th ioester intermediates in the Family I mechanism. This may be possible to confir m using tritiated sodium borohydride as has been done for Family I enzymes.66 NaBH4 has been used to reduce mixed anhydrides of carboxylic acids and carbonic acids,67 and cyclic carboxylic acid anhydrides.68 Figure 3-9. Stereo picture s howing the interactions of the oxalyl-aspartyl anhydride with residues in the FRC dimer. The letter designation (A or B) in the numbering scheme indicates the FRC monomer in which the residue is located. Obtaining protein crystals of FRC with bound formyl-CoA or oxalyl-CoA proved elusive due to the lability of the thioesters. Surprisingly, after extensive screening studies, the product of reaction between FRC and oxalyl-CoA, i.e. the putative oxalylAsp169 anhydride intermediate (Figures 3-8 and 3-9), was crystallized by Stefano Ricagno, a graduate student in the laborator y of Dr. Ylva Lindqvist the crystallography collaborator.64 This structure lends very strong support to the partic ipation of mixed

PAGE 57

46 anhydrides of Asp169 in the catalytic mechanis m of FRC, and along with the kinetic data presented in Chapter 2 strongly supports the proposed ordered Bi Bi mechanism shown in Figure 3-8.

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47 CHAPTER 4 EXPERIMENTAL All materials were of the highest purity available and, unless stated otherwise, obtained from Fisher Scientific Internati onal, Inc. (Hampton, NH) or Sigma-Aldrich Corp. (St. Louis, MO). Protein concentra tions were determined by the Lowry method as modified by Hartree69 using bovine serum albumin as a standard. DNA sequencing was performed by the DNA Sequencing Core of the Interdisciplinary Center for Biothechnology Research at the University of Florida. The BL21(DE3) Escherichia coli expression strain transformed with pET-9a plasmid carrying the gene for wild-type FRC was supplied by Dr. Harmeet Sidhu (Ixion Biot echnology, Inc., Alachua, FL). Mutagenic PCR (polymerase chain reaction) primer s were obtained from GenoMechanix LLC (Gainesville, FL) (D169A and D169E) and Integrated DNA Technologies, Inc. (Coralville, IA) (D169S). Expression and Purification of FRC Wild-type formyl-CoA transferase (FRC) from Oxalobacter formigenes was overexpressed in BL21(DE3) Escherichia coli and purified by anion exchange and affinity chromatography. The enzyme was expressed by growing an overnight culture (100 mL) in Luria-Bertani broth containi ng 30 g/mL kanamycin (LBK) at 37C and 200-250 rpm, which was then used to inoculate 4-6 L of fresh LBK. FRC expression was induced by adding IPTG (isopropyl--D-thiogalactopyranoside) wh en the optical density of the culture, grown at 37C and 200 rpm, r eached 0.6-0.8 at 600 nm (4 mL of 0.1 M IPTG added per liter of culture). The cells were harvested 2.5-4.0 hours after induction

PAGE 59

48 by centrifugation at 4C. The cell pellets were resuspended in 50-150* mL lysis buffer (100 mM potassium phosphate, 1 mM dithiothre itol (DTT), pH 7.2) and lysed by passing two times through a French press or by soni cation (ten 10 second pul ses with 30 second intervals). After centrifuging, the lysate supernatant was loaded on a 120 mL DEAE Fast Flow anion exchange column equilibrated with buffer A (25 mM sodium phosphate, 1 mM DTT, pH 6.2) and FRC eluted by stepping to 35% buffer B (25 mM sodium phosphate, 1.0 M NaCl, 1 mM DTT, pH 6.2) at 5 mL/min. The FRCcontaining fractions were loaded on a 20 mL Blue FF (Blue SepharoseTM 6 Fast Flow) affinity column equilibrated with buffer A. The column was then washed with 50% buffer B and FRC finally eluted with buffer C (25 mM glycine, 1 mM DTT, 20% isopropanol, pH 9.0) at 4 mL/min. The FRC-containing fractions from the affinity column were immediately buffer-exchanged by passing through a 135 mL G-25 desalting column equilibrated with buffer A at 10 mL/min. The protein solution was then injected on a 60 mL Q-Sepharose high performance anion exchange (QHP) column equilibrated with buffer A and the FRC eluted at 5 mL/min by stepping to 20% bu ffer B for 1-2 column volumes followed by a linear gradient to 35%B over 67 column volumes, with FRC eluting close to the center of the linear gradient. Glycer ol was added to achieve a conc entration of 10% to stabilize the protein since it has a te ndency to precipitate. The re sulting protein solution was stored at -80C. All purification steps we re run at ambient temperature and the FRC fractions stored on ice between purification st eps. The purification was verified by SDSPAGE with Coomassie Blue staining and act ivity measurements of the fully purified enzyme. See Appendix A for typical FPLC chromatograms from each purification step. Smaller volumes used when lysing with French press. Larger volumes used when lysing by sonication.

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49 The pooled FRC-containing fractions after the first three chromatography steps have a tendency to form a protein precipit ate, resulting in loss of CoA transferase activity. This loss is minimized by using 1 mM dithiothreitol (DTT) in all buffers and storing the collected fractions on ice until the ne xt purification step. For best yields, the first three steps should be performed within 12 hours. The fully purified FRC eluted off the QHP column also has a slight tendency to precipitate, but addition of glycerol to a concentration of 10% stabilizes the protein so no precipitate is vi sible after freezing and thawing. The Blue FF affinity column re sin has a lifetime of about one year when storage instructions are followed and usi ng a column that has started to degrade dramatically decreases yields. Expression of Selenomethionine Derivative of Wild-Type FRC The selenomethionine derivative of wild -type FRC (SeMet FRC) was prepared using literature procedures for expression of SeMet proteins in nonauxotrophic strains of E. coli.51 The bacteria were grown in M9 medium (2 mM MgSO4, 0.1 mM CaCl2, 48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 4 g/L glucose) at 37C and 200 rpm until the optical density of the culture at 600 nm reached 0.6-0.8. Methionine biosynthesis by the bacteria wa s then downregulated by addi tion of lysine (100 mg/L), threonine (100 mg/L), phenylalanine (100 mg/L ), leucine (50 mg/L), isoleucine (50 mg/L), valine (50 mg/L), and proline (50 mg /L). Selenomethionine (50 mg/L) was then added to the culture, which was incubated fo r 15 minutes before inducing with IPTG as described for wild-type FRC above. The cel ls were harvested 45 hours after induction and the SeMet FRC purified as described for wild-type FRC.

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50 Site-Directed Mutagenesis Mutagenic primers were designed using Ge ne Runner v. 3.05 (Hastings Software, Inc.). The pET-9a plasmid with the FRC gene insert was isolated from BL21(DE3) E. coli with the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI) and used as a template for PCR with the mutagenic primers using the QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA). The desired mutations were verified by DNA sequencing of the FRC gene inserts of the mutated pET-9a plasmids isolated from transformed XL-1 or XL-10 Go ld supercompetent cells (Stratagene, La Jolla, CA). BL21(DE3) competent cells (N ovagen, Madison, WI) were then finally transformed with the mutated plasmids and the mutated FRC proteins expressed and purified as described for wild-type FRC above. Synthesis of CoA Esters Formyl-CoA Formylthiophenyl ester was ma de by formylating thiophenol53 using a formylating reagent made from acetic anhydride and formic acid.54 Formic acid (6.9 g; 150 mmols; 5.8 mL) was added dropwise to stirred acetic anhydride (7.7 g; 75 mmols; 7.1 mL) and the resulting mixture stirred at 45 C for 2.5 hours. Pyridine (59 mg; 0.75 mmols; 61 L) was then added, immediately followed by thi ophenol (5.5 g; 50 mmols; 5.1 mL), and the reaction mixture stirred at room temperatur e for 24 hours and then stored at 7 C overnight after purging with dry nitrogen gas. Thin layer chromatography (TLC) (1:1 chloroform:hexanes) showed the product at Rf = 0.53 and no reactant (Rf = 0.72) with a faint spot at Rf = 0. The remaining acids and anhydrides were removed from the reaction mixture by vacuum distillation (40 – 50C/20 mmH g) and collected in a cold-finger trap immersed in liquid nitrogen. A water aspirator was used, connected via a drying tube to

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51 the reaction flask. When the volume had d ecreased to 6-8 mL the product was washed with an equal amount of deionized wate r and then dried over anhydrous magnesium sulfate. TLC, after washing with water, indicated minor decomposition of the product. The product was finally vacuum distilled ( 115-117 C/23 mmHg), yielding 3.1 g or 45 % of pure product. The literature values are 101 C/15 mmHg and 87 % yield. 1H-NMR (CDCl3, 300 MHz) 10.22 (s, 1), 7.46 (m, 5). 13C-NMR (CDCl3, 75.5 MHz) 190.0, 134.1, 129.9, 129.5, 126.0. See NMR spectra in Appendix B. Formyl-CoA was prepared by a method based on the procedure by Sly and Stadtman.52 Sodium salt of CoA (80 mg, 96 mols CoA) was dissolved in 2-8 mL of icecold deionized water and the pH adjusted to 7.0 with 0.1 M KOH or NaOH while cooling on ice. Formylthiophenyl ester (80 mg, 580 mol s) was dissolved in ice-cold dry ether (1-2 mL) and added quickly to the cold Co A solution while stirring, resulting in pH decrease to 6.6. The pH was ad justed to 7.5-8.0 with 1 M KHCO3 or NaHCO3 at pH 8.0 (0.25-1.0 mL) and the reaction mixture stirred on ice for 2-3 minutes. The pH was then carefully adjusted to 3.0 with 0.1 M HCl under rapid stirring, a nd the solution washed three times with two volumes of ice-cold ether. The pH was finally adjusted to 5.5 with 0.1 M KOH or NaOH and stored at -80C. Typical yields were about 70-90%. LC/(+/ )ESI-MS (Flow Injection Analysis) calculated for C22H36N7O17P3S: 795.5; found: 795.1 (+)ESI-MS and 795.6 ( )ESI-MS. Oxalyl-CoA Thiocresol oxalate (25 mg, 0.13 mmol) diss olved in ice-cold anhydrous ether (5-10 mL) was added slowly to an ice-cold solu tion of CoA (sodium sa lt) (25 mg, 0.031 mmol) in water (10 mL) at pH 7.5 (adjusted with 0.1 M NaHCO3). The reaction mixture was stirred on ice for about 20 min before re moving the aqueous layer and acidifying it

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52 carefully to pH 3.5 with 0.11.0 M HCl fo llowed by washing with two 12 mL portions of ice-cold ether. When complete removal of thiocresol oxalate was desired the aqueous phase was washed eight times with ether adju sting the pH of the aqueous phase to 3.0 between washes. The resulting solution was stored at -80 C Typical yields were 80100%. C-18 HPLC/UV(260 nm)/(+)ESI-MS calculated for C23H36N7O19P3S: 839.5; found: 839.4. Analysis of CoA Esters Analysis of the CoA esters wa s based on literature procedures57 using C-18 reversed-phase HPLC (Dynamax Microsor b 60-8 C18, 250 x 4.6 mm) with a singlewavelength detector at 260 nm. The column was equilibrated with 86% buffer A (25 mM NaOAc, pH 4.5) and 14% buffer B (20 mM NaOAc, pH 4.5, 20% CH3CN) running at 1.0 mL/min. Immediately after injection a 12 mi nute gradient to 34% buffer B was started followed by a step to 100% buffer B for 2 minut es before returning to 14 % buffer B. Oxalyl-CoA eluted at 6.5 minutes, free CoAS H at 11.5 minutes, and formyl-CoA at 12.5 minutes. The CoA esters were quantitated by integration of thei r peaks in the HPLC chromatograms using free CoASH as a quantita tive standard. The va lidity of this method was confirmed for formyl-CoA by independently measuring its concentration in solution using the hydroxylamine method,58-60, and for oxalyl-CoA by measuring the concentration of oxalate in an oxalyl-CoA solution before and after complete hydrolysis by base using an oxalate detection kit (Sigma-Aldrich Corp., St. Louis, MO). Purification of CoA Esters The CoA esters were purified using a preparative C-18 reversed-phase column (Dynamax 60A C18, 250 x 21.4 mm). The column was equilibrated with 88% mobile phase A (10 mM sodium phosphate at pH 5.0) and 12 % mobile phase B (mobile phase A

PAGE 64

53 with 20% acetonitrile) at 10 mL /min. Two minutes after injecting the sample the fraction of mobile phase B was increased linearly to 38% over 17 minutes. The absorbance of the eluent at 260 nm was monitored and fracti ons collected manually on ice. Oxalyl-CoA eluted at about 5 min, CoA at 12 min, and formyl-CoA at 13 min. The fractions were analyzed as described above and lyo philized in small (1-2 mL) aliquots.* Enzymatic Assay The recombinant wild-type FRC was assayed by measuring the initial rate of oxalyl-CoA formation by HPLC analysis of quenched aliquots. The assay mixture contained 60 mM potassium phosphate (pH 6.7), FRC (90 ng 9.5 nM), and variable amounts of substrates and inhibitors (if de sired) in a total volume of 200 L. The reaction was started by addition of formyl-CoA after incubating the other components at 30 C for about 30 seconds. Aliquots of th e reaction mixture (90 L) were typically taken after 60 s and 90 s and quenched with 10% acetic acid** (10 L) before quantitating oxalyl-CoA by reversed-phase HPLC using a s horter version of the analytical procedure described above that separate s only oxalate and oxalyl-CoA from the rest of the mixture components.64 No formation of oxalyl-CoA was de tected in control experiments when the enzyme or either substrate was omitted, or when FRC denatured by incubation in boiling water was used. The limit of detection of the assay as described is about 0.05 M of oxalyl-CoA when 75 L of quenched assa y mixture is injected on HPLC column. The specific activities of the D169A, D169E, and D169S FRC mutants were assayed using an identical procedure except th at reaction mixtures were incubated for up Small aliquots minimized hydrolysis of the CoA esters during freeze-drying. ** The pH of the quenched aliquots should be 3-4, which is ideal for CoA ester stability. Oxalyl-CoA concentration in quenched samples remains unchanged for several hours when stored on ice.

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54 to 60 minutes prior to quenching. In additi on, because of the much lower activity of the FRC mutants, the amount of enzyme in each as say was increased to 2 g, and the initial concentrations of oxalate and formyl-C oA were 100 mM and 200 M, respectively. Alternative substrates (Table 2-3) were screened using the a ssay described above, replacing the natural substrates with the substrates being tested. CoA ester concentrations were in the range of 80-200 M and free acid concentrations were 62.5125 mM in these experiments. Equilibrium Constant Determination Wild-type FRC (18 g) was incubated at 22C with 73 M formyl-CoA, 50 M oxalate, 13 M formate, and 13 M free CoA (introduced by formyl-CoA stock solution) in 60 mM potassium phosphate buffer at pH 6.7 (200 L total volume). Aliquots (45 L) were withdrawn after 10, 27, and 52 minutes, a nd quenched with 10% acetic acid (5 L). The concentrations of oxalyl-CoA, free CoA (and therefore formate), and formyl-CoA in each sample was measured by HPLC as described above. Equilibrium concentrations were reached after 27 minutes, giving Keq = 32 3. Size Exclusion Chromatography (SEC) A BIOSEP SEC-S2000 column (300 x 7.8 mm with 75 x 7.8 mm guard column) (Phenomenex, Torrance, CA) was equilibrate d with 100 mM potassium phosphate at pH 6.6 running at 1.0 mL/min. The column was calibrated using lysozyme (14.4 kDa), carbonic anhydrase (29.0 kDa), peroxidase (44.0 kDa), bovine serum albumin (66.0 kDa), alcohol dehydrogenase (150 kDa), and -amylase (200 kDa). The void volume of the column was measured by injecting blue de xtran. Samples of FRC (7 g and 30 g) were injected on the column and the mo lecular weights (53.8 kDa and 81.0 kDa) calculated from the retention times.

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55 CHAPTER 5 SUMMARY This research project was originally ai med at determining whether the mechanism of FRC was a Ping-Pong mechanism, used by almost all known CoA transferases known at the time. However, upon discovering th at FRC belongs to a new class of CoA transferase enzymes, apparently using a nove l mechanism of CoA transfer, the focus of the project turned to deciphering that mechan ism. The unique crystal structure of FRC and sequence alignments of Family III Co A transferase enzymes identified a putative main catalytic residue, which was confirmed by its mutation and the resulting loss of activity, thus refuting a mechanism like the on e used by Family II CoA transferases as proposed previously.25 Following synthesis of substr ates and development of an enzymatic assay, steady-state kinetic studies and product inhibition patterns led to the proposal of a novel mechanism of CoA tran sfer, which includes covalent enzymesubstrate anhydride intermediate s. One of these putative intermediates was observed by X-ray crystallography of FRC crystals grown in the presence of oxa lyl-CoA, providing further evidence for the propos ed mechanism. Since the main catalytic residue is conserved in known Family I II CoA-transferases, the cata lytic mechanism of formylCoA transferase is almost certainly employed by all other members of this enzyme family. Although no direct evidence for the proposed covalent enzyme-substrate anhydride intermediates exist for Family III CoA transferases the crystal structure data presented herein are all supportive of such intermediates. An 18O-exchange experiment using 18O-

PAGE 67

56 oxalate with formyl-CoA or 18O-formate with oxalyl-CoA should provide definitive evidence for these intermediates (Figure 3-8) as it has for Family I CoA transferases.32 So far, no convenient proteolytic conditions, which are necessary for such experiments, have been found for FRC. The possibility of a covalent enzyme-CoA thioester in the mechanism of FRC, as observed for Family I CoA transferases (F igure 1-3A), can be tested by using the methods of Hersh and Jencks.33 After incubation of the enzyme with a CoA ester substrate and subsequent removal of sma ll molecules by size exclusion filtration the presence of free CoA can be assayed after allowing the putative enzyme-CoA intermediate to hydrolyze. In cubation of the enzyme with free CoA serves as a control reaction, since no CoA should be detected in the protein fraction af ter the size exclusion filtration. Some other questions remain regarding the proposed mechanism, such as the timing of the mixed anhydride formation, whic h could take place before or after oxalate binding, although the observation of the aspart yl-oxalyl anhydride in termediate in the crystal structure would suggest this happens in the absence of the free acid substrate, since there was no oxalate or formate present in the solution the crystals were grown in. It is not known whether CoA stays bound afte r the mixed anhydride formation or if it diffuses into solution. In theory, at least th e pantotheine moiety may have to move out of the active site to allow the incoming free acid to attack the mixed anhydride, unless the attack comes from the other side of the anhydride intermediate. One of the biggest questions concerns the substrate selectivity of FRC. The features of the active site responsible for the high selectiv ity towards formate, oxalate, and succinate, while acetate,

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57 oxamate, and pyruvate are not accepted as substrates, are not yet known. Perhaps computational modeling can provide some answers to these questions, identifying favorable or unfavorable interactions be tween active site residues and the bound molecules. More site-directed mutagenesis studies aimed at residues in the active site known or suspected of interacting with the bound substrates, such as Gln17, Tyr59, and Tyr139, would also undoubtedly be benefici al to the understanding of the mechanism. Another issue is th e discovery of the yfdW gene in E. coli and the expression of its protein product, whose crystal stru cture is the same as FRC’s. E. coli has never been shown to degrade oxalate, although the presence of this gene and the orthologous gene of oxalyl-CoA decarboxylase from O. formigenes would indicate that it is capable, given that these proteins have the same function as in O. formigenes. An interesting experiment would be to examine whether E. coli can be induced to express these genes and become an active oxalate degrader. Furthermore, comparing the activities and substrate specificities of the enzymes from th ese two sources would be of value. Given that FRC and oxalyl-CoA decarboxyl ase (OXC) combined constitute up to 20% of the total protein content of O. formigenes,11,13 and the important function they serve in the cell, it is not unreasonable to suggest that these enzymes may interact somehow, perhaps creating oxalate-degrading co mplexes inside the cell. This would be beneficial to the organism, especially sin ce it would minimize spont aneous hydrolysis of the labile formyl-CoA, which would effectively stop the catalytic cycle of oxalate breakdown. The fact that the adenine part of CoA is firmly bound to FRC while the rest of the molecule is solvent accessible coul d mean that the pantetheine arm of CoA esterified with oxalate can swi ng out of the active site of FRC directly into the active site

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58 of OXC and return to FRC as a formyl ester after decarboxylation. This hypothesis could be tested by fluorescence labeling and/ or immunohistochemistry methods. Finally, understanding the folding mechanis m of the remarkable interlocked dimer of FRC could be a large contribution to the fi eld of protein folding, and collaboration in that regard is currently under way.

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59 APPENDIX A PROTEIN PURIFICATION CHROMATOGRAMS The following figures show typical chroma tograms of the four purification steps used to purify the wild-type, selenomethion ine, and mutated form yl-CoA transferases. Manual Run 1:1_UV Manual Run 1:1_Cond Manual Run 1:1_Conc 0 500 1000 1500 2000 2500 3000 mAu 0 100 200 300 400 500 600 700 ml Figure A-1. FPLC chromatogram of the DEAE anion exchange purification step. Blue = Absorbance at 280 nm (0-3000 mAu*). Red = Conductivity (scale 0-75 mS/cm). Black = Percent concentration of buffer B (scale 0-100%). The arrow points to the FRC containing peak. mAu = milli absorbance unit

PAGE 71

60 Manual Run 1:1_UV Manual Run 1:1_Cond Manual Run 1:1_Conc 0 500 1000 1500 mAu -50 0 50 100 150 200 250ml Figure A-2. FPLC chromatogram of the Bl ueFF affinity purification step. Blue = Absorbance at 280 nm (0-1700 mAu). Red = Conductivity (scale 0-45 mS/cm). Black = Percent concentration of buffer B (scale 050%). The arrow points to the FRC peak. Manual Run 1:1_UV Manual Run 1:1_Cond 0 100 200 300 400 500 600 700 mAu 0 50 100 150 ml Figure A-3. FPLC chromatogram of the bu ffer exchange purification step. Blue = Absorbance at 280 nm (0-750 mAu). Red = Conductivity (scale 0-22 mS/cm).

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61 Manual Run 5:1_UV Manual Run 5:1_Cond Manual Run 5:1_Conc 0 20 40 60 80 100 mAu 0 100 200 300 400 500 600 700 ml Figure A-4. FPLC chromatogram of the QHP anion exchange purification step. Blue = Absorbance at 280 nm (0-120 mAu). Red = Conductivity (scale 0-75 mS/cm). Black = Percent concentration of buffer B (scale 0-100%). The arrow points to the FRC peak.

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62 APPENDIX B NMR SPECTRA OF FORMYLTHIOPHENYL ESTER The following figures in show the 1H-NMR and 13C-NMR spectra of formylthiophenyl ester, the formylating r eagent used to prepare formyl-CoA. See experimental section (Chapter 4) for chemical shifts. Figure B-1. 1H-NMR spectrum of formylthiophenyl ester.

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63 Figure B-2. 13C-NMR spectrum of formylthiophenyl ester. Figure B-3. Close-up on rele vant peaks of Figure A-2.

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64 LIST OF REFERENCES (1) Hodgkinson, A. Oxalic Acid in Biology and Medicine; Academic Press: London, 1977. (2) Kotsira, V. P.; Clonis, Y. D. Arch. Biochem. Biophys. 1997, 340, 239-249. (3) Aguilar, C.; Urzua, U.; Koenig, C.; Vicuna, R. Arch. Biochem. Biophys. 1999, 366, 275-282. (4) Shimazono, H. J. Biochem. 1955, 42, 321-340. (5) Tanner, A.; Bornemann, S. J. Bacteriol. 2000, 182, 5271-5273. (6) Khindaria, A.; Grover, T. A.; Aust, S. D. Arch. Biochem. Biophys. 1994, 314, 301306. (7) Urzua, U.; Kersten, P. J.; Vicuna, R. Appl. Environ. Microbiol. 1998, 64, 68-73. (8) Urzua, U.; Kersten, P. J.; Vicuna, R. Arch. Biochem. Biophys. 1998, 360, 215-222. (9) Quayle, J. R.; Keech, D. B.; Taylor, G. A. Biochem. J. 1961, 78, 225-236. (10) Quayle, J. R. Biochem. J. 1963, 87, 368-373. (11) Baetz, A. L.; Allison, M. J. J. Bacteriol. 1990, 172, 3537-3540. (12) Quayle, J. R. Biochem. J. 1963, 89, 492-503. (13) Baetz, A. L.; Allison, M. J. J. Bacteriol. 1989, 171, 2605-2608. (14) Allison, M. J.; Dawson, K. A.; Mayberry, W. R.; Foss, J. G. Arch. Microbiol. 1985, 141, 1-7. (15) Cornick, N. A.; Allison, M. J. Can. J. Microbiol. 1996, 42, 1081-1086. (16) Cornick, N. A.; Yan, B.; Bank, S.; Allison, M. J. Can. J. Microbiol. 1996, 42, 1219-1224. (17) Cornick, N. A.; Allison, M. J. Appl. Environ. Microbiol. 1996, 62, 3011-3013.

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65 (18) Duncan, S. H.; Richardson, A. J.; Kaul, P.; Holmes, R. P.; Allison, M. J.; Stewart, C. S. Appl. Environ. Microbiol. 2002, 68, 3841-3847. (19) Williams, H. E.; Wandzilak, T. R. J. Urol. 1989, 141, 742-747. (20) Sidhu, H.; Schmidt, M. E.; Cornelius, J. G.; Thamilselvan, S.; Khan, S. R.; Hesse, A.; Peck, A. B. J. Am. Soc. Nephrol. 1999, 10, S334-S340. (21) Sidhu, H.; Hoppe, B.; Hesse, A.; Tenbrock K.; Bromme, S.; Rietschel, E.; Peck, A. B. Lancet 1998, 352, 1026-1029. (22) Sidhu, H.; Allison, M. J.; Chow, J. M.; Clark, A.; Peck, A. B. J. Urol. 2001, 166, 1487-1491. (23) Kumar, R.; Mukherjee, M.; Bhandari, M.; Kumar, A.; Sidhu, H.; Mittal, R. D. Eur. Urol. 2002, 41, 318-322. (24) Breslow, R. J. Am. Chem. Soc. 1958, 80, 3719-3726. (25) Heider, J. FEBS Lett. 2001, 509, 345-349. (26) Leutwein, C.; Heider, J. Microbiol.-UK 1999, 145, 3265-3271. (27) Leutwein, C.; Heider, J. J. Bacteriol. 2001, 183, 4288-4295. (28) Dickert, S.; Pierik, A. J.; Buckel, W. Mol. Microbiol. 2002, 44, 49-60. (29) Dickert, S.; Pierik, A. J.; Linder, D.; Buckel, W. Eur. J. Biochem. 2000, 267, 38743884. (30) Elssner, T.; Engemann, C.; Baumgart, K.; Kleber, H. P. Biochemistry 2001, 40, 11140-11148. (31) Engemann, C.; Elssner, T.; Kleber, H. P. Arch. Microbiol. 2001, 175, 353-359. (32) Selmer, T.; Buckel, W. J. Biol. Chem. 1999, 274, 20772-20778. (33) Hersh, L. B.; Jencks, W. P. J. Biol. Chem. 1967, 242, 3481-3486. (34) Pickart, C. M.; Jencks, W. P. J. Biol. Chem. 1979, 254, 9120-9129. (35) Buckel, W.; Dorn, U.; Semmler, R. Eur. J. Biochem. 1981, 118, 315-321. (36) Mack, M.; Buckel, W. FEBS Lett. 1995, 357, 145-148. (37) Buckel, W.; Buschmei.V; Eggerer, H. H.-S. Z. Physiol. Chem. 1971, 352, 11951205. (38) Dimroth, P.; Eggerer, H. P. Natl. Acad. Sci. USA 1975, 72, 3458-3462.

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66 (39) Buckel, W.; Bobi, A. Eur. J. Biochem. 1976, 64, 255-262. (40) Sidhu, H.; Ogden, S. D.; Lung, H. Y.; Luttge, B. G.; Baetz, A. L.; Peck, A. B. J. Bacteriol. 1997, 179, 3378-3381. (41) Gruez, A.; Roig-Zamboni, V.; Valencia, C.; Campanacci, V. R.; Cambillau, C. J. Biol. Chem. 2003, 278, 34582-34586. (42) Ricagno, S.; Jonsson, S. ; Richards, N.; Lindqvist, Y. EMBO J. 2003, 22, 32103219. (43) Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 104-137. (44) Segel, I. H. In Enzyme Kinetics; Wiley Classics Library Edition ed.; WileyInterscience: New York, 1993; pp 274-345. (45) Cleland, W. W. In The Enzymes; 3 ed.; Boyer, P. D., Ed.; Academic Press: New York, 1970; Vol. 2, pp 1-65. (46) Segel, I. H. In Enzyme Kinetics; Wiley Classics Library Edition ed.; WileyInterscience: New York, 1993; pp 560-665. (47) Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 173-187. (48) Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 188-196. (49) Studier, F. W.; Rosenberg, A. H.; Dunn, J. J.; Dubendorff, J. W. Method. Enzymol. 1990, 185, 60-89. (50) Studier, F. W. J. Mol. Biol. 1991, 219, 37-44. (51) Doublie, S. Method. Enzymol. 1997, 276, 523-530. (52) Sly, W. S.; Stadtman, E. R. J. Biol. Chem. 1963, 238, 2632-2638. (53) Bax, P. C.; Stevens, W. Recl. Trav. Chim. Pay.-B. 1970, 89, 265-269. (54) Stevens, W.; Van Es, A. Recl. Trav. Chim. Pay.-B. 1964, 83, 863-872. (55) Stolle, R. Ber. Deut. Chem. Ges. 1914, 47, 1130-1132. (56) Schutte, H.; Flossdorf, J.; Sahm, H.; Kula, M. R. Eur. J. Biochem. 1976, 62, 151160. (57) Demoz, A.; Garras, A.; Asiedu, D. K.; Netteland, B.; Berge, R. K. J. Chromatogr. B 1995, 667, 148-152. (58) Lipmann, F.; Tuttle, L. C. J. Biol. Chem. 1945, 159, 21-28.

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67 (59) Stadtman, E. R. Method. Enzymol. 1957, 3, 228-231. (60) Sly, W. S.; Stadtman, E. R. J. Biol. Chem. 1963, 238, 2639-2647. (61) White, H.; Jencks, W. P. J. Biol. Chem. 1976, 251, 1688-1699. (62) Segel, I. H. In Enzyme Kinetics; Wiley Classics Library Edition ed.; WileyInterscience: New York, 1993; pp 767-783. (63) Ricagno, S.; Jonsson, S. ; Richards, N.; Lindqvist, Y. Acta Crystallogr. D 2003, 59, 1276-1277. (64) Jonsson, S.; Ricagno, S.; Li ndqvist, Y.; Richards, N. G. J. J. Biol. Chem. 2004, in press. (65) Moore, S. A.; Jencks, W. P. J. Biol. Chem. 1982, 257, 10882-10892. (66) Hersh, L. B.; Jencks, W. P. J. Biol. Chem. 1967, 242, 339-340. (67) Soai, K.; Yokoyama, S.; Mochida, K. Synthesis-Stuttgart 1987, 647-648. (68) Nose, A.; Kudo, T. Yakugaku Zasshi 1975, 95, 1390-1396. (69) Hartree, E. F. Anal. Biochem. 1972, 48, 422-427.

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68 BIOGRAPHICAL SKETCH Stefn Jnsson was born in Reykjavk, Icel and, in 1972. He holds a B.Sc. degree in biochemistry from the University of Icel and and an M.Sc. degree in chemistry from the same school. After working one year as a rese arch scientist for deC ODE Genetics, Inc in Reykjavik he started graduate studies in the Chemistry Department of the University of Florida in August 1999, where he joined Dr. Ni gel G. J. Richards research group in May 2000.


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Title: The Mechanism of Formyl-Coenzyme A Transferase, a Family III CoA Transferase, from Oxalobacter formigenes
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Material Information

Title: The Mechanism of Formyl-Coenzyme A Transferase, a Family III CoA Transferase, from Oxalobacter formigenes
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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THE MECHANISM OF FORMYL-COENZYME A TRANSFERASE, A FAMILY III
COA TRANSFERASE, FROM Oxalobacterformigenes















By

STEFAN JONSSON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































This dissertation is dedicated to my family.















ACKNOWLEDGMENTS

This study was supported by grants from the National Institutes of Health

(DK61666 and DK53556) and by the University of Florida Chemistry Department.

Partial funding was also received from Dr. Ammon B. Peck.

Thanks go to my doctoral dissertation committee: Dr. Steven A. Benner, Dr.

Ronald K. Castellano, Dr. Ammon B. Peck, Dr. Jon D. Stewart, and especially my

advisor, Dr. Nigel G. J. Richards, for making this research project a reality.

Stefano Ricagno and Dr. Ylva Lindqvist, I thank for fruitful collaboration and their

excellent crystallographic work.

I am grateful to my coworkers and friends in the Richards research group,

especially Dr. Christopher H. Chang for proofreading, countless discussions and valuable

insights, Drazenka Svedrumi for her help, and Sue Abbatiello for her mass spectrometry

efforts, and Dr. Jianqiang Wang for supplying thiocresol oxalate monoester.

I thank Dr. James A. Deyrup, Graduate Coordinator, and Lori Clark in the Graduate

Student Office for their help and advice.

Dr. Harmeet Sidhu, Ixion Biotechnology, Inc., I thank for providing the original

expression strain ofE. coli, training, and for use of equipment and resources at Ixion

during the summer of 2000.

Finally, I thank my family for supporting me, especially my wife, Aslaug

Hognad6ttir, for all of her support and love throughout my graduate studies and for

proofreading.















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES ....................................................... ............ ....... ....... vi

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. vii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

O x alic A cid B reak dow n ............................................................... .......................... 1
C oenzym e A Transferases .................................................. .............................. 3
O v erview ................................................................... 3
Form yl-C oA Transferase........................................... ............... ...............5.
K inetics of B isubstrate Enzym es ........................................................ ............... 8
O v e rv ie w ............................................................................................................... 8
Initial Velocity Studies .................. ...................... ............... .... ...........
Product Inhibition Studies .............................................................. ... ............ 11
R research Objective ..................................................... ................. 13


2 EXPRESSION, PURIFICATION, AND KINETIC STUDIES OF FORMYL-COA
TRAN SFERASE .................................... .. ......... .......... .... 14

Expression and Purification of Recombinant Formyl-CoA Transferase................14
Synthesis of CoA Esters ... ...... .......................................................... 16
F orm yl-C oA .......................................................................16
Oxalyl-CoA .............. .... ...................... 17
Purification ................ ........ .....................17
Stability .................................................. 18
A ssay D evelopm ent ......... .... .. .............................. ............... 19
E quilibrium C onstant........... ...... .......................................................... .... .... .... .. 23
Alternative Substrates ......... ..... ........ .. .. ......... ........ ............... 23
K inetics ............. ....................... ........................................25
Initial R ates........................................................ 26
Product Inhibition............................................. 28
Inhibition by formate.............. .. ..................................... 29
Inhibition by oxalyl-CoA ............... ......................... ............... 31









Inhibition by Coenzym e A ........................................................................... 32


3 STRUCTURE AND MECHANISM OF FORMYL-COA TRANSFERASE ...........33

S tru c tu re ...................................... ................................. ................ 3 3
A ctiv e S ite ................................................................3 6
Point M station Studies .................................. .. .. ...... ............ 39
M ech anism ......................................... ....................................................... 42


4 E X PE R IM E N T A L ......................................... .. .. ...................................................47

Expression and Purification of FRC ............................ ...................... .................47
Expression of Selenomethionine Derivative of Wild-Type FRC.............................49
Site-D directed M utagenesis............................................................... .....................50
Synthesis of C oA E sters ..................................................................... ..................50
F orm y l-C oA ................................................................50
O x a ly l-C o A ................................................................................................... 5 1
A analysis of C oA E sters .............................................. ............................. 52
Purification of C oA E sters........................................................ ............... 52
E nzym atic A ssay .................. .......................................... .............. .. .... ...... 53
Equilibrium Constant D eterm nation .................................. .................. ............... 54
Size Exclusion Chrom atography (SEC) ........................... .......................................54


5 SU M M A R Y ...............................................................................................55



APPENDIX

A PROTEIN PURIFICATION CHROMATOGRAMS ........................ .................59

B NMR SPECTRA OF FORMYLTHIOPHENYL ESTER ........................................62

L IST O F R E F E R E N C E S ............................ ................................................................64

B IO G R A PH IC A L SK E TCH ..................................................................... ..................68













v
















LIST OF TABLES


Table pge

1-1. Previously reported properties of native wild-type formyl-CoA transferase from
Oxalobacterform genes. ................................................ ................................ 6

2-1. Purification table of FRC showing typical yield and purification level ..................15

2-2. Effect of excessive dilution of the enzyme stock solution on specific activity.........22

2-3. Summary of alternative substrates screening. ................................ .................24

2-4. Kinetic constants of FRC calculated from initial rates data (Figures 2-9 and 2-10). 26

2-5. Product inhibition patterns observed for various mechanisms of bisubstrate
e n z y m e s .......................................................................... 2 8

2-6. Inhibition constants for format and oxalyl-CoA...................................................28
















LIST OF FIGURES


Figure page

1-1. Currently recognized classes of enzymes that catalyze the direct or indirect
degradation of oxalate. ..................................... ........................ 1

1-2. Structure of Coenzym e A ........................................ ................................. 4

1-3. Previously known enzyme-catalyzed mechanisms of CoA transfer .......................4

1-4. Crystal structure of the interlocked FRC dimer with bound CoA..............................7

1-5. Cleland notation of the three main types of Bi Bi mechanisms. ............................9

1-6. Ordered Bi Bi mechanism showing the individual rate constants (top), and the
equation for the initial forward velocity in the absence of products (bottom). .......10

1-7. Lineweaver-Burk plots of initial velocity data for a bisubstrate enzyme..................11

1-8. Lineweaver-Burk plots showing inhibition patterns in enzyme kinetics. .................12

2-1. SDS-PAGE gels showing the expression level and purification of FRC..................15

2-2. Synthetic schemes for formyl-CoA and oxalyl-CoA. .............................................16

2-3. Three HPLC chromatograms showing the hydrolysis of oxalyl-CoA by KOH........ 17

2-4. Three HPLC chromatograms showing extraction of thiocresol oxalate monoester
from oxalyl-CoA reaction mixture. ........................................ ....................... 18

2-5. Graphs for determining the rate of hydrolysis of the CoA esters..............................19

2-6. HPLC chromatograms of assay mixture aliquots quenched after 5 min (A), 15 min
(B ), and 55 m in (C ). ......................................... ... .... ................. 20

2-7. Linear increase of initial rate with increasing enzyme concentration .....................22

2-8. Structures of free acids and ester parts of CoA esters used for alternative substrate
screening (see T able 2-3). ............................................................. .....................25

2-9. Lineweaver-Burk plot of initial rates at three fixed formyl-CoA concentrations. ....27









2-10. Lineweaver-Burk plot of initial rates at four fixed oxalate concentrations.............27

2-11. Lineweaver-Burk plot showing product inhibition by format vs. formyl-CoA at
fixed unsaturating oxalate concentration............... ........... ............ .............. 29

2-12. Lineweaver-Burk plot showing product inhibition by format vs. formyl-CoA at
fixed saturating oxalate concentration...........................................................30

2-13. Lineweaver-Burk plot showing product inhibition by format vs. oxalate at fixed
unsaturating formyl-CoA concentration........................ ....................... 30

2-14. Lineweaver-Burk plot showing product inhibition by oxalyl-CoA vs. oxalate at
fixed unsaturating formyl-CoA concentration. ................................... ............... 31

2-15. Lineweaver-Burk plot showing inhibition by CoA vs. oxalate at fixed unsaturating
form yl-C oA concentration. ........................................................... .....................32

3-1. Monomer and homodimer structures of recombinant formyl-CoA transferase. .......34

3-2. Size exclusion chromatography data used to calculate the molecular mass of FRC.35

3-3. Size exclusion HPLC chromatograms of FRC ............................................ 35

3-4. Stereo picture of Coenzyme A in its binding site............... .... .................36

3-5. CLUSTAL W (1.82) multiple sequence alignment of Family III CoA transferases.37

3-6. Stereo images of active site structures of FRC mutants completed with CoA.........40

3-7. Stereo picture of the end of the pantetheine chain of CoA and amino acids in the
surrounding active site. ........................... .......... ......... ........... 42

3-8. Proposed mechanism of formyl-CoA transferase from Oxalobacterformigenes. ....43

3-9. Stereo picture showing the interactions of the oxalyl-aspartyl anhydride with
residues in the FR C dim er. .............................................. .............................. 45

A-1. FPLC chromatogram of the DEAE anion exchange purification step. ..................59

A-2. FPLC chromatogram of the BlueFF affinity purification step. ..............................60

A-3. FPLC chromatogram of the buffer exchange purification step. ............................60

A-4. FPLC chromatogram of the QHP anion exchange purification step......................61

B- 1H-NM R spectrum of formylthiophenyl ester. ......................... ............... ......62

B-2. 13C-NM R spectrum of formylthiophenyl ester. .............................. ............... .63









B-3. Close-up on relevant peaks of Figure A-2. ............. .......................... ............... 63















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE MECHANISM OF FORMYL-COENZYME A TRANSFERASE, A FAMILY III
COA TRANSFERASE, FROM Oxalobacterformigenes

By

Stefan J6nsson

December 2004

Chair: Nigel G. J. Richards
Major Department: Chemistry

Formyl-Coenzyme A transferase (FRC) is a part of an oxalate-degrading catalytic

cycle in Oxalobacterformigenes, a bacterium which colonizes the gastrointestinal tract of

many mammals, including humans, symbiotically degrading toxic oxalate ingested with

food and produced as a byproduct of normal cellular metabolism. FRC is a member of a

recently recognized Family III CoA transferases, which apparently use a novel

mechanism of CoA transfer as indicated by the limited kinetic studies that have been

published so far.

FRC from 0. formigenes was overexpressed in Escherichia coli and purified by

anionic exchange and affinity chromatography. The selenomethionine derivative of FRC

was also expressed and purified, allowing determination of the X-ray crystal structure of

the enzyme. Mutations of the putative main catalytic residue, identified by analysis of

the crystal structure of FRC with bound CoA, caused very diminished or complete loss of

transferase activity. Steady-state initial rate kinetic studies on the wild-type enzyme









indicate a ternary complex (sequential) mechanism rather than Ping-Pong kinetics, which

are observed for the well known Family I CoA transferases, and product inhibition

studies strongly support an ordered Bi Bi mechanism. A catalytic mechanism is

proposed, based on the crystal structure and kinetic data, where the main catalytic residue

forms mixed anhydrides with format and oxalate during catalytic turnover. One of these

proposed intermediates, an aspartyl-oxalyl anhydride, was observed in a crystal structure

obtained from crystals of the wild-type FRC grown in the presence of oxalyl-CoA,

lending further evidence to the proposed mechanism.



















CHAPTER 1
INTRODUCTION


Oxalic Acid Breakdown


Oxalic acid is a byproduct of normal cellular metabolism and is toxic to almost all


organisms.1 Several oxalate-degrading processes have therefore naturally evolved


(Figure 1-1):


O
(A) 0 0
0


0
O


O
(B) 0 0
O


Oxalate oxidase
02,2H+




Oxalate decarboxylase
cat. 02 H+


02

(C) 2 HO2C202- 2 [(HO2C202-)Mn3]+2 4 CO2


2 Mn+3 < Manganese peroxidase H20 + 2 Mn2

2 H20 2 H+


0
(D) O SCoA
0


0
(E) 0 +
0


Oxalyl-CoA reductase



NAD(P)H NAD(P)+


SCoA
H )SCoA


0

0 H
0


CoAS-


O
Formyl-CoA transferase 0 SCoA
SCoA
o


0

H O


OxalHl-CoA decarboxlasCO
Oxalyl-CoA decarboxylase


Figure 1-1. Currently recognized classes of enzymes that catalyze the direct or indirect
degradation of oxalate in (A) plants and fungi, (B) fungi and bacteria, (C)
fungi only, and (D, E) bacteria only.


2CO, + HO,


0

H -o









* (A) Oxalate oxidase, found mainly in plants2 and more recently in fungi,3 catalyzes
the oxidation of oxalate to carbon dioxide with concomitant reduction of dioxygen
to hydrogen peroxide.

(B) Oxalate decarboxylase, found mainly in fungi4 and more recently in bacteria,5
decarboxylates oxalate in the presence of catalytic amounts of dioxygen.

(C) Manganese peroxidase, secreted by some fungi, catalyzes the formation of
Mn+3 from Mn+2 and hydrogen peroxide. Oxalate-Mn+3 complexes then
spontaneously form and break down to Mn+2, carbon dioxide and hydrogen
peroxide in the presence of dioxygen.6-8

(D) Oxalyl-CoA reductase, also known as glyoxylate dehydrogenase, found in
bacteria able to use oxalate as a source of carbon (oxalotrophic bacteria), catalyzes
the NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dependent
reduction of oxalyl-CoA to glyoxylate and free CoA.9'10

(E) Formyl-CoA transferase (FRC)11 and oxalyl-CoA decarboxylase (OXC),12'13
also found in oxalotrophic bacteria, form a catalytic cycle that breaks down oxalate
to carbon dioxide and format via formyl- and oxalyl-CoA ester intermediates.

Bacterial enzymes are the least studied of the oxalate-degrading enzymes. Of particular

interest are FRC and OXC, which have been purified and characterized from Oxalobacter

formigenes, an anaerobic Gram-negative bacterium, which colonizes the gastrointestinal

tract of many warm-blooded animals, including humans.11'13'14 0. formigenes is unique

since oxalate is the only compound that supports its growth, although small amounts of

acetate are also required.14 Approximately 99% of the oxalate consumed by the

bacterium is decarboxylated to CO2 and format by OXC and FRC (Figure 1-1E) and the

rest is used for cell biosynthesis, presumably through the action of oxalyl-CoA reductase

(Figure 1-1D).14-17 OXC and FRC activities are therefore crucial for the bacterium's

survival and play a central role in its oxalate metabolism. Absence of this bacterium

from human intestinal flora has been strongly linked to pathological conditions that can

arise if oxalate accumulates in the human body, including hyperoxaluria (increased levels

of oxalate in urine), the formation of kidney stones (urolithiasis), renal failure,









cardiomyopathy, and cardiac conductance disorders.18-23 OXC is a TPP-dependent

(thiamine pyrophosphate-dependent) decarboxylase.13 The mechanisms of TPP-

dependent enzymes are generally well known24 and will not be discussed here. The

amino acid sequence of FRC, however, bears no similarity to the well known Family I

CoA transferases or Family II CoA transferases. Instead, FRC, by sequence similarity,

belongs to a newly recognized Family III CoA transferases (Pfam accession number

PF02515),25 for which only limited kinetic studies11'26-31 and no mechanistic studies have

been reported until now.

Coenzyme A Transferases

Overview

The reversible transfer of the CoA moiety (Figure 1-2) from CoA thioesters to free

acids is catalyzed by CoA transferases (Figure 1-1E) which were, until recently, grouped

into two enzyme families. The well known Family I contains CoA transferases for 3-

oxoacids, short-chain fatty acids, and glutaconate. The transfer reaction proceeds via a

Ping-Pong mechanism, where a glutamate residue of the enzyme forms covalently bound

anhydrides and CoA thioesters during catalysis.32 These enzymes incorporate 180 when

18s-containing CoA acceptor is used in the reaction since one oxygen atom is transferred

from the incoming free acid to the main catalytic residue during one catalytic cycle

(Figure 1-3A). Family I CoA transferases are inactivated by incubation with

hydroxylamine or sodium borohydride (NaBH4) in the presence of CoA thioesters.32-36

Family II includes only the homodimeric a-subunits of citrate lyase and citramalate

lyase, which catalyze the transfer of an acyl carrier protein (ACP) containing a covalently

bound CoA derivative. In a ternary complex mechanism a direct attack of the incoming

citrate or citramalate on the acetyl thioester of the acetylated CoA derivative results in the











formation of a mixed anhydride of acetate and citrate or citramalate during catalytic


turnover (Figure 1-3B). No covalent enzyme-substrate intermediates are formed in the

mechanic 37-39
mechanism.


H H
I I
( N N,-SH
Pantetheine SH
'OH 0
P0 0
Pyrophosphate 0 O 0 N
OH OH

OO
0
P 0
HO-P\
OH


N> Adenine


S Ribose-3-phosphate

OH


Figure 1-2. Structure of Coenzyme A.


I
Glu 0











Glu 0


R2 SCo


0
O

R A-

ACP-CoAS


Glu O RI Glu SCoA


,A -SCoA







Glu Ok R2 Glu jSCoA

-SCoA
'A


0 0

R O

ACP-CoAS-


0 R


0



R 0

R 0

O

R2 x0-


0

0 0SCoA-ACP

R SCoA-ACP


Figure 1-3. Previously known enzyme-catalyzed mechanisms of CoA transfer. (A) Ping-
Pong mechanism of Family I CoA transferases. Oxygen atoms from the
incoming free acid are shown in bold font. (B) Ternary complex mechanism
of Family II CoA transferases.

The new Family III of CoA transferases currently includes only three characterized


enzymes other than formyl-CoA transferase from 0. formigenes (FRC). These are BbsF


(A)




0

R2 SCoA


0

RI SCoA









(succinyl-CoA: (R)-benzylsuccinate CoA transferase from Thauera aromatica),26'27 FldA

((E)-cinnamoyl-CoA: (R)-phenyllactate CoA transferase from Stickland-fermenting

Clostridia),28'29 and CaiB (butyrobetainyl-CoA: (R)-carnitine CoA transferase from E.

coli and Proteus sp.).30,31 The subunits of these enzymes have similar masses, 42-47

kDa, while their quaternary structures include homodimers (FRC and CaiB), 2322

aggregates (BbsF), and a subunit in a heterotrimeric enzyme complex (FldA).

Additionally, there are many putative proteins that have been identified as probable

Family III CoA transferases based on DNA and/or translated amino acid sequence

similarities. A ternary mechanism with no covalent enzyme-substrate intermediates,

similar to the mechanism of Family II CoA transferases, has been proposed for Family III

enzymes based on the limited kinetic data reported that indicate a sequential mechanism

rather than Ping-Pong.25

Formyl-CoA Transferase

Unmodified native wild-type FRC has 428 amino acids. The monomeric mass is

47.3 kDa and its pi is 5.2 calculated from the amino acid sequence.40 FRC catalyzes

transfer of the CoA moiety from formyl-CoA to oxalate producing oxalyl-CoA and

format (Figure 1-1E). Previous literature on this enzyme is limited to earlier studies on

the oxalate-degrading Pseudomonas oxalaticus,9'12 which were the first indication of the

existence of such an enzyme, and more recently the purification and limited

characterization of FRC isolated from Oxalobacterformigenes,11 the cloning of the FRC

gene from 0. formigenes and subsequent overexpression of the recombinant FRC in

Escherichia coli.40 In addition, a gene (yfdW) from E. coli sharing 61% sequence identity

with FRC has been cloned and expressed as a His-tagged gene product.41 It is not yet

known whether the yfdW gene product is an active transferase. There are no reports of









oxalate degradation by E. coli, and it is not known if the yfdW gene is expressed under

standard growth conditions or what factors could induce its expression.

Table 1-1. Previously reported properties of native wild-type formyl-CoA transferase
from Oxalobacterformigenes. One unit (U) equals one micromole of CoA
transferred per minute.11,40
Properties Values / Observations
Km (formyl-CoA) vs. excess succinate 3.0 + 0.5 mM
Vmax (formyl-CoA) vs. excess succinate 30 U/mg
Km (oxalate) vs. formyl-CoA 5.1 0.5 mM
Vmax (oxalate) vs. formyl-CoA 6.4 U/mg
Km (succinate) vs. formyl-CoA 2.3 0.6 mM
Vmax (succinate) vs. formyl-CoA 19 U/mg
Maximum specific activity for transfer of 2.2 U/
CoA from formyl-CoA to oxalate
pH optimum 6.5 7.5
Isoelectric point (calculated) 5.2
Isoelectric point (experimental) 4.7
Molecular weight (calculated) 47.3 kDa
Molecular weight (experimental) 44.7 kDa (active as monomer)
Acceptable CoA acceptors Oxalate, succinate
Not acceptable CoA acceptors Acetate, malonate
Acceptable CoA donors Formyl-CoA, succinyl-CoA
Not acceptable CoA donors Acetyl-CoA
Inhibition 20% by 1.0 mM N-ethylmaleimide
91% by 1.0 mM p-chloromercuribenzoate
i10 mM EDTA, 10 mM Ca+2
No inhibition +2
10 mM Mg 1.0 mM TPP

In the previous characterization the enzyme was assayed with a continuous,

coupled enzyme assay in which format dehydrogenase (FDH) was used to detect the rate

of format production by monitoring the formation of NADH (reduced form of

nicotinamide adenine dinucleotide) spectrophotometrically. Findings of the previous

characterization of the native wild-type FRC are summarized in Table 1-1.11 Although

formyl-CoA and succinyl-CoA can both act as CoA donors, there is no report of oxalyl-









CoA acting as one, so there is so far no evidence of FRC being able to catalyze the

reverse reaction (transfer of CoA from oxalyl-CoA to formate.






















Figure 1-4. Crystal structure of the interlocked FRC dimer with bound CoA. For clarity,
the protein monomers are colored red and green and represented by molecular
ribbons. The bound CoA molecules are shown as space-filling models, which
are colored using the following scheme: H white; C grey; N blue; O red;
S yellow; P purple.

The structure of recombinant FRC from 0. formigenes has recently been published

and represents the first crystal structure of a member of the new Family III CoA

transferases.42 The structure is a novel fold where two circular shaped monomers are

interlocked like two links in a chain creating a tightly packed homodimer. Each dimer

has two active sites located on opposite sides of the structure in a cleft between the

monomers (Figure 1-4). The structure, with bound coenzyme A molecules, allowed

D169 to be identified as the most likely main catalytic residue (see Chapter 3). The yfdW

gene from E. coli yields a protein with the same structure.41









Kinetics of Bisubstrate Enzymes

Overview

The majority of enzymes catalyze reactions between two or more substrates

yielding two or more products. Enzymes that use two substrates and yield two products,

such as FRC, employ one of several possible Bi Bi mechanisms (using Cleland's

nomenclature where Bi Bi refers to two substrates and two products).43 The three main

forms of Bi Bi mechanisms are (A) sequential random Bi Bi, where substrates bind in

random order and products are released in random order, (B) sequential ordered Bi Bi,

where substrates bind in an ordered manner and products are released in a specific order,

and (C) Ping-Pong Bi Bi, where the first product is released before binding of the second

substrate (Figure 1-5).43 The sequential mechanisms are also known as ternary complex

mechanisms. Some variations of these general scenarios exist, but are not relevant to this

discussion.

Furthermore, the above systems can be under either rapid equilibrium conditions or

steady-state conditions. Rapid equilibrium conditions refer to when all binding and

dissociation steps are very rapid compared to the catalytic step, and the rate-limiting step

is the breakdown of EAB to E + P + Q. This describes some random binding systems

well, but the steady-state approach is preferred for sequential ordered and Ping-Pong

mechanisms.44 The steady-state approach describes systems where the isomerization of

the central complex (EAB) and product release are so rapid that E, EA, and EAB never

attain equilibrium, but are kept at near-constant, or steady-state levels (Figure 1-5). This

is generally the case when the substrate concentrations and the values of Km (Michaelis

constant) for the substrates greatly exceed the enzyme concentration.43'45'46









A B


EAB EPQ


E EA EAB EPQ EQ E

(C)
A P B Q

1 I l t
E EA FP F FB EQ E

Figure 1-5. Cleland notation of the three main types of Bi Bi mechanisms. (A) Random,
(B) ordered, and (C) Ping-Pong. A and B are the substrates, E is the free
enzyme, F is a stable enzyme intermediate, and P and Q are the products. The
two and three letter species are transitory (unstable) complexes.43

Initial Velocity Studies

The first step in determining the kinetic mechanism of an enzyme is usually initial

velocity studies. Initial velocity refers to the rate of the catalyzed reaction in the absence

of products. Generally, the reaction is said to be at initial velocity when less than 10% of

the substrates have been used and product formation is still linear. Exceptions from this

rule include cases where the equilibrium constant for the catalyzed reaction is very small

or one of the products is removed from the assay mixture, for example if the product is

CO2. The velocity data are usually plotted in double reciprocal plots (Lineweaver-Burk

plots) as 1/v vs. 1/[A] or 1/[B] with the other substrate at a fixed concentration. Velocity

equations for multisubstrate enzymes have been derived and the microscopic rate

constants (ki, k -1, etc.) grouped into kinetic constants (Vmax, Km, etc.) that can be

determined by experiments. The ordered Bi Bi mechanism, for example, has ten

individual rate constants that can be combined in such a way that the initial forward








velocity in the absence of products can be described by an equation containing only four

kinetic constants (Figure 1-6).43'45'46
k, k4
E + A EA+ B EQ + P E + Q
-1 k -4

k -2 k2 k3 k -3

kp
EAB EPQ
k-p

Sk3k4kp 1 kl
V..= [E], -
max k3k+ k3k + k4kp + k4k a k, '

Km k3k4kp k4 (k2k3 + k-2kp + k3kp)
mA k, (k3k4 + k3k + k4k + k4k) mB k2 (k3k4 + k3kp + k4k, + k4kp)

v [A][B]
Vma KKB + KmB [A] + K [B] + [A][B]

Figure 1-6. Ordered Bi Bi mechanism showing the individual rate constants (top), and
the equation for the initial forward velocity in the absence of products
(bottom).43,46

The double reciprocal forms of the velocity equation from Figure 1-6 are
1 Km KamB 1 1I mB
S 1 + + 1 + when [A] is varied and [B] is constant,
v Vmax KmA [B] [A] Vmax [B]

and

1 Km K 1 1 Km
B 1 + a-- 1 + A when [B] is varied and [A] is constant.
v Vmax [A] [B] Vma [A]

Therefore, except in the rare case when Kia is very small compared to KmA, the data

should give intersecting lines, since both the slope and intercepts of the 1/v vs.

1/[substrate] plots are affected by the value of the constant substrate concentration. The









lines will intersect above, on, or below the x-axis, depending on the ratio of KmA/Kia

(Figure 1-7A).46

The random Bi Bi mechanism gives more complex equations describing nonlinear

double reciprocal plots unless one substrate is saturating, and equations for the Ping-Pong

mechanism describe parallel lines in the double reciprocal plots (Figure 1-7B).43'46

(A) (B)
l/v 1/v




Increasing [B]
Increasing [B]creasing [B]


1/[A] 1/[A]

Figure 1-7. Lineweaver-Burk plots of initial velocity data for a bisubstrate enzyme. (A)
Intersecting lines indicating a random or ordered sequential ternaryy complex)
mechanism. (B) Parallel lines suggesting Ping-Pong kinetics. The same
patterns appear when the other substrate is varied.

Product Inhibition Studies

Taking the reverse of the catalyzed reaction into consideration, complete velocity

equations have been derived and written for A or B as the varied substrate in the presence

of P or Q. The effects of P and Q on the slopes and intercepts of the lines in double

reciprocal plots can be read from the equations and the type of inhibition predicted.

Three main types of inhibitors are recognized in enzyme kinetics: (A) Competitive

inhibitors only affect the slope, but not the 1/v-axis intercept, (B) uncompetitive

inhibitors only affect the intercept, but have no effect on the slope, and (C) mixed-type

inhibitors (also known as noncompetitive inhibitors) affect both the slope and intercept

(Figure 1-8).43,46-48









(A) 1/v (B) 1/v (C) 1/v
Increasing [I] Increasing [I] Increasing [I]




1/[Substrate] 1/[Substrate] 1/[Substrate]

Figure 1-8. Lineweaver-Burk plots showing inhibition patterns in enzyme kinetics. (A)
Competitive inhibition, (B) uncompetitive inhibition, and (C) mixed-type
(noncompetitive) inhibition. For product inhibitions I is either P or Q and [A]
is varied at constant [B] or vice versa.

The complete velocity equation for the ordered Bi Bi mechanism, rearranged to

show inhibition by P vs. A, at constant [B] is

v _[A]
Vmax KlaKm Km [P] KmB KmQ [ [P
K 1+ 1+ + [A] 1+ 1+ + [p]
mA KmA [B] K K m [ B K Kmp K
J iq mp V q mP
Slope factor Intercept factor

where the indicated slope and intercept factors will be factors in the slope and intercept of

the reciprocal equation. Therefore, at low (unsaturating) [B], the inhibition by P is a

mixed-type inhibition since both the slope and intercept factors are a function of [P]. At

very high (saturating) [B], the slope factor approaches unity, but the intercept factor is

still dependent on [P], so the inhibition is uncompetitive.46-48

Rearranged to show inhibition by P vs. B, at constant [A], the equation becomes

v _[B]
Vmax K I la + K [P] I KmA + P]
K 1 + 1 + + [Bj 1 + A
mB [A] Kq Kmp [A] KA
Slope factor Intercept factor

showing that P is a mixed-type inhibitor at all concentrations of A, since very high

(saturating) [A] leaves [P] terms in both slope and intercept factors.46-48








Inhibition by Q vs. A, at constant [B] is described by

v [A]
Vmax Q KK K
K 1 + + mB + [A] 1 + m
mA K, KmA [ B] B
Slope factor Intercept factor

which shows that Q is a competitive inhibitor at all concentrations of B, since [Q] has an

effect on the slope factor at any [B], but has no effect on the intercept factor.46-48

Finally, the inhibition by Q vs. B, at constant [A] is described by

v [B]
Vmax K U \Q] KmA( \QI]
Ka 1+ Ka 1 + Q + [B] 1+ KmA 1+
mB [A] K [A] K
Slope factor Intercept factor

showing that Q is a mixed-type inhibitor at relatively low (unsaturating) concentrations of

A with both the slope and intercept factors being functions of [Q]. At very high [A]

(saturating) there is no inhibition since the [Q] terms are eliminated from both factors.46-48

Velocity equations for other possible Bi Bi mechanisms show different product

inhibition patterns, making it possible to distinguish between mechanisms solely by

product inhibition studies.46-48 Some examples are shown in Chapter 2 (Table 2-5).

Research Objective

The main objective of this project was to obtain a thorough understanding of the

mechanism of CoA transfer by formyl-CoA transferase, a key enzyme in oxalate

breakdown by Oxalobacterformigenes. A mechanism is proposed based on kinetic and

crystallographic data (Figure 3-8). This is the first detailed mechanistic study of a Family

III CoA transferase, and suggests a novel mechanism of CoA transfer likely employed by

all members of this class of enzymes.














CHAPTER 2
EXPRESSION, PURIFICATION, AND KINETIC STUDIES OF FORMYL-COA
TRANSFERASE

The results of kinetic studies and all requirements for the enzyme kinetics are

covered in this chapter, including protein expression, protein purification, synthesis of

thioester substrates, and enzyme assay development.

Expression and Purification of Recombinant Formyl-CoA Transferase

FRC was produced by IPTG (isopropyl-/f-D-thiogalactopyranoside) induction of

Escherichia coli, carrying the FRC gene in a pET-9a vector. This bacterial strain,

commonly used for protein expression, has the advantage of being deficient in proteases

that might degrade the desired protein, and it carries a gene for T7 RNA polymerase,

whose transcription is controlled by the IPTG inducible lacUV5 promoter.49'50 Upon

induction by IPTG the bacterium starts producing T7 RNA polymerase which recognizes

a T7 promoter located shortly upstream of the FRC gene in the pET-9a plasmid. Thus

the bacterium starts producing FRC after addition of IPTG.

The purification of FRC was based on a procedure developed by Kjell Eriksson and

Billi Herzer (Fast Trak process development, Amersham Pharmacia Biotech, Inc.,

currently known as Amersham Biosciences) for Ixion Biotechnology, Inc (personal

communication). The enzyme was purified from crude lysate of harvested cells by two

steps of anion exchange, one step of affinity chromatography, and a buffer exchange step.

Various adjustments were made to the original procedure to optimize the yield of active

enzyme. The enzyme proved stable enough to do all the FPLC (fast performance liquid









chromatography) work at ambient temperature if it was kept on ice between column runs,

and the first three purification steps were performed on the same day. The yield of

purified FRC was typically 10-15 mg of highly pure protein from each liter of culture

(Table 2-1 and Figure 2-1). See Appendix A for examples of FPLC chromatograms of

the FRC purification.

Table 2-1. Purification table of FRC showing typical yield and purification level. Some
activity measurements were not reliable due to precipitation of protein in the
samples prior to assaying, and are omitted from the table.
Total Specific Total Yield Purification
protein activity activity (%U) (fold)
Purification step (mg) (U/mg) (U)
Cell lysate 1150 0.51 587 100
DEAE anion exchange 333 -
BlueFF affinity chromatography 67
and buffer exchange
Q Sepharose High Performance
( P63 6.11 385 66 12
(QHP) anion exchange


1 5 6


7 8 9 10 11 12


*- -~ ~ q


RFR


q
.- .,


Figure 2-1. SDS-PAGE gels showing the expression level and purification ofFRC. (A)
1. Cell pellet at time of induction; 2. Cell pellet 150 minutes after induction; 3.
Molecular weight markers (kDa). (B) 4. Pooled FRC fractions from DEAE
column; 5. Empty lane; 6. BlueFF flowthrough fractions; 7. BlueFF low salt
wash; 8. MW markers (same as lane 3A); 9. Pooled FRC fractions after buffer
exchange; 10. BlueFF NaOH wash; 11. QHP low salt wash; 12. Fully purified
FRC from QHP. Both gels were stained with Coomassie Blue.

The selenomethionine derivative of FRC, which was needed to solve the protein

crystal structure, was prepared by inhibiting the natural biosynthesis of methionine by the









expression strain grown in minimal medium, and adding selenomethionine before IPTG

induction.51 The selenomethionine derivative was purified as described above for the

recombinant wild-type FRC. Mass spectrometry showed full incorporation of

selenomethionine.

No formyl-CoA transferase activity was detected in a cell lysate of expression

strain cells, containing a pET-9a plasmid without the FRC gene, grown under the same

conditions as when FRC was expressed. FRC was therefore assumed to be responsible

for all the CoA transferase activity isolated from the expression strain.

Synthesis of CoA Esters

The CoA ester substrate and product were synthesized using a thioester exchange

reaction between CoA and an aromatic thiol ester of the appropriate acid (Figure 2-2).

(A) O 0
H-O O

45C, 2.5 hrs

0 0
SSH H O 'S H CoASH
S cat. pyridine / 0 pH 8 CoAS H

(B)
So- CoASH CoAS
/ S pH 8


Figure 2-2. Synthetic schemes for formyl-CoA and oxalyl-CoA. A) Synthesis of formyl-
CoA from CoA and formylated thiophenol. B) Synthesis of oxalyl-CoA from
thiocresol oxalate and CoA.

Formyl-CoA

Formyl-CoA was produced by allowing CoA to react with an excess of

formylthiophenyl ester as described by Sly and Stadtman.52 The formylthiophenyl ester

was made by formylating thiophenol53 using a formylating reagent made from acetic









anhydride and formic acid as described by Stevens and van Es54 (Figure 2-2A). The

formylation of CoA was verified by mass spectrometry, and the expected change in

retention time on reverse phase C-18 column by HPLC was observed (peaks 3 and 4 in

Figure 2-6).

Oxalyl-CoA

Oxalyl-CoA was synthesized by a similar method, except the precursor was

thiocresol oxalate monoester made previously by Dr. Jianqiang Wang by the method of

Stolle55 (Figure 2-2B). The synthesis was verified by mass spectrometry and by HPLC

(Figure 2-3).

1 (A) 1 (B) 2 (C)






2

3 3





Figure 2-3. Three HPLC chromatograms showing the hydrolysis of oxalyl-CoA by
KOH. (A) Crude oxalyl-CoA reaction mixture. (B) Same as (A), but spiked
with CoA. (C) Same as (A), but after hydrolysis with KOH. Peak
assignments: 1 Oxalyl-CoA; 2 CoA; 3 Thiocresol oxalate monoester; 4 -
Thiocresol. The chromatograms show absorbance at 260 nm vs. time and all
have same or similar scale. Peaks were assigned by coinjecting standards.
The slight discrepancy in retention times of peak 3 in (A) and (B) is due to
instrumentation problems.

Purification

The excess aromatic thiol esters were removed from the crude reaction mixtures by

multiple ether extractions, adjusting the pH of the aqueous phase to 3.0 prior to each









extraction (Figure 2-4). Both CoA esters were further purified by preparative reverse

phase HPLC followed by freeze-drying to minimize the amount of CoA in their stock

solutions.

(A) 1 () (C)









2
rAA-







Figure 2-4. Three HPLC chromatograms showing extraction of thiocresol oxalate
monoester from oxalyl-CoA reaction mixture. (A) Crude reaction mixture.
(B) After four extractions with ether. (C) After eight extractions with ether.
Peak assignments: 1 Oxalyl-CoA; 2 Thiocresol oxalate monoester. The
chromatograms show absorbance at 260 nm vs. time and all have same or
similar scale. Peaks were assigned by coinjecting standards. The slight
discrepancy in retention times of peak 2 in (A) and (B) is due to
instrumentation problems.

Stability

Stabilities of the CoA esters under the assay conditions were determined by

measuring the pseudo first-order rate for the uncatalyzed hydrolysis of these compounds

at pH 6.7 and 300C. The concentrations of the CoA esters were measured by HPLC as

described below. The half-lives were calculated from the pseudo first-order rate

constants taken as the slope of the best-fit line of the plots in Figure 2-5, as described by

the first-order rate equation: ln[A] = ln[A]0 kt. This gave an estimate of 150 min for

the half-life of formyl-CoA, which is in reasonable agreement with a literature value of

300 min in aqueous solution at room temperature and neutral pH.52 The measured half

life for oxalyl-CoA was about 10 days, which is also consistent with previous reports that










solutions of oxalyl-CoA at pH 6.5 are stable for weeks when stored at -15C.10 The large

difference in the rate of uncatalyzed hydrolysis of the two thioesters can likely be

attributed to the presence of the negatively charged carboxylate group in oxalyl-CoA,

which will destabilize the tetrahedral adduct formed by nucleophilic attack of water on

the thioester carbonyl.


4.6 (A) -4.60- (B)


4.5 -
S 11/2 t 150 min t 10 days
S4.5910 days
4.4


4.3 4.58
0 20 40 60 0 100 200 300 400
t(min) t (min)


Figure 2-5. Graphs for determining the rate of hydrolysis of the CoA esters. (A) Formyl-
CoA and (B) oxalyl-CoA.

Assay Development

The only other previous study on FRC used a coupled enzyme assay to determine

the rate of format production by monitoring the formation of NADH from NAD+ by

format dehydrogenase (FDH).11 A more direct approach of determining the rate of

oxalyl-CoA formation by HPLC was developed for the studies described here. The FDH

coupled assay consistently produced lower rate values than the HPLC-based assay. Since

NAD+ was suspected to inhibit FRC due to its structural similarity to the CoA esters, the

effect of 0.5 and 1.0 mM NAD+ in the HPLC assay was studied. Rapid hydrolysis of

formyl-CoA was observed and very little production of oxalyl-CoA was detected. The

hydrolysis of formyl-CoA in the presence of NAD+ may explain the high value of Km for

formyl-CoA (3.0 mM) published in the original study of FRC.11 This value is closer to









the Km value of format for the FDH used (13 mM),56 than the published Km values for

CoA esters in two other Family III CoA transferases (3 and 40 aM).27'29

4

1 2
2

(A)







(B) .00------







(C) o ....

Figure 2-6. HPLC chromatograms of assay mixture aliquots quenched after 5 min (A),
15 min (B), and 55 min (C). Peak assignments: 1 Oxalate; 2 Oxalyl-CoA;
3 CoA; 4 Formyl-CoA. The chromatograms show absorbance at 260 nm
vs. time and all have same or similar scale. Peaks were assigned by injecting
standards.

The facile separation of CoA and its esters by reverse phase HPLC57 is the basis for

the enzymatic assay used in the work described here. The rate of oxalyl-CoA production

by the enzyme can be determined from the amount of oxalyl-CoA present in assay

mixtures quenched at different timepoints as demonstrated in Figure 2-6. Similar

conditions were used as in the previous study, except no other enzyme reaction was

coupled to the one being studied. The enzyme, substrates, and inhibitors when

appropriate, were added to phosphate buffer at pH 6.7, adding formyl-CoA last to initiate

the reaction. Aliquots of the assay mixture were quenched with acetic acid at two









different timepoints, typically after 1.0 and 1.5 minutes, stored on ice, and analyzed by

HPLC within 60 minutes.

The efficacy of the quench was verified by performing the assay at pH 3-4 instead

of pH 6.7. No oxalyl-CoA had formed after 28 min, nor after overnight incubation at

ambient temperature. Furthermore, the formyl-CoA peak area did not decrease

appreciably over the first 28 min, but had dropped somewhat overnight due to hydrolysis

as evidenced by an equal increase in the CoA peak area. After quenching, the pH is

between 3 and 4, which is favorable for the stability of CoA esters, thus there was no

detectable decrease in oxalyl-CoA or formyl-CoA content in quenched samples stored on

ice for up to 60 minutes.

Free CoA and CoA esters were quantitated by HPLC using a standard solution of

CoA. The assumption was made that the extinction coefficients of the CoA esters at 260

nm, the detection wavelength, were the same as for CoA. This assumption was

successfully validated for formyl-CoA by measuring its concentration in solution using

the hydroxylamine method,58-60 and for oxalyl-CoA by measuring the concentration of

oxalate in an oxalyl-CoA solution before and after hydrolysis by base using an oxalate

oxidase-based detection kit (Sigma-Aldrich Corp., St. Louis, MO).

The rate of the catalyzed reaction increased linearly with increasing enzyme

concentration (Figure 2-7). Diluting the enzyme stock solution excessively before

assaying, however, resulted in lowered specific activity (Table 2-2). This most likely

resulted from dissociation of the homodimeric enzyme into its subunits upon dilution, a

proposal supported by size exclusion chromatography studies discussed in Chapter 3. A

20-fold dilution of a 0.90 mg/mL stock enzyme solution retained its specific activity well










over a few days when stored at 40C, while 40-fold and 80-fold diluted solutions lost their

specific activity rapidly. Typically the 20-fold diluted stock solution lost only about 15

% of its transferase activity when stored at 40C for 3 weeks.

25 -

20 -

I 15

d 10 -

5 -

0
0 10 20 30 40 50 60
[FRC] (pg/mL)


Figure 2-7. Linear increase of initial rate with increasing enzyme concentration.

Table 2-2. Effect of excessive dilution of the enzyme stock solution on specific activity.
Protein concentration of the original stock was 0.90 mg/mL. The same
amount of enzyme was used in all assays.
Dilution (fold) Storage time at 0-40C (hours) Relative specific
before assaying activity
20X 1 1.0
20X 24 1.0
40X (made from 20X) 1 0.8
40X (made from 1X stock) 24 0.6
80X (made from 40X) 1 0.4
80X (made from 20X) 0 0.9
80X (made from 20X) 1 0.8
80X (made from 20X) 3 0.4

No transferase activity was detected when one of the substrates or the enzyme was

not present in the assay mixture, and no activity was detected when enzyme denatured by

incubation in boiling water for 5 minutes was used.

The detection limit of the HPLC assay depends on the amount of enzyme in the

assay mixture, and the length of incubation. Long incubation times are not desirable for

initial rate measurements due to the liability of formyl-CoA, but can be used if the goal is









merely to see if there is some measurable activity or not, such as when screening for

alternative substrates or checking mutant enzymes for transferase activity.

Equilibrium Constant

The equilibrium constant for the FRC-catalyzed CoA transfer was estimated by

measuring the equilibrium concentrations of formyl-CoA and oxalyl-CoA incubated with

FRC and known initial amounts of format and oxalate. The value of Keq was

determined to be 32 + 3, favoring oxalyl-CoA and format to formyl-CoA and oxalate.

This value is similar to the equilibrium constant determined for the reaction catalyzed by

succinyl-CoA:acetoacetate transferase.61

Alternative Substrates

Results of the former study identifying succinate and succinyl-CoA as alternative

substrates, but not acetyl-CoA,11 were confirmed and a variety of other potential

substrates were screened. Somewhat surprisingly, the natural substrates, formyl-CoA and

oxalate, did not yield the highest rate of CoA transfer. The highest specific activities

were observed for CoA transfer from succinyl-CoA to format and from formyl-CoA to

succinate. CoA transfer from formyl-CoA to glutarate was also faster than to oxalate,

while CoA transfer to maleate was slower. Malonyl-CoA, methylmalonyl-CoA, acetyl-

CoA, and propionyl-CoA were not used as CoA donors by FRC with oxalate or format

as acceptors. Propionate, oxamate, and pyruvate were not accepted as substrates (Figure

2-8 and Table 2-3).

Acetate did not inhibit the enzyme at concentrations of up to 60 mM, while acetyl-

CoA inhibited the rate of CoA transfer by 25% at 100 [tM and 45% at 200 [tM

concentration in the presence of 35 pM formyl-CoA and 100 mM oxalate. There was no

indication of an irreversible inhibition by acetyl-CoA, since the enzyme was preincubated










with the inhibitor before adding formyl-CoA. Acetyl-CoA therefore appears to bind to

FRC but the thioester is not lysed by the enzyme. This is not surprising since the

homologous protein from E. coli has been crystallized with bound acetyl-CoA.41 The

remaining alternative substrates were not assayed for inhibition, but it seems likely that

any CoA ester will inhibit the enzyme by competing for the CoA binding site with the

natural substrate even if the thioester moiety cannot be used as substrate.

Table 2-3. Summary of alternative substrates screening. CoA ester concentrations were
80-200 [tM and free acid concentrations were 62.5-125 mM.
CoA Donor Acceptor (free acid) Approx. specific activity*
(ptmol/min.mg)
Formyl-CoA Oxalate 5.5 (forward reaction)
Oxalyl-CoA Formate 0.7 (reverse reaction)
Succinyl-CoA Oxalate 4.5
Succinyl-CoA Formate 40
Formyl-CoA Succinate 50
Formyl-CoA Glutarate 15
Formyl-CoA Maleate 2
Formyl-CoA Oxamate 0
Formyl-CoA Pyruvate 0
Formyl-CoA Acetate 0
Oxalyl-CoA Acetate 0
Malonyl-CoA Formate 0
Malonyl-CoA Oxalate 0
Methylmalonyl-CoA Formate 0
Methylmalonyl-CoA Oxalate 0
Acetyl-CoA Formate 0
Acetyl-CoA Oxalate 0
Propionyl-CoA Formate 0
Propionyl-CoA Oxalate 0
*The limit of detection was approximately 0.001 gmol/min*mg

The overall selectivity rule for CoA transfer by FRC based on the observations

above appears to be a requirement for a negative charge on the thioester end of the

incoming CoA ester substrate, and the free acid substrate needs to be a dianion. The only

exceptions to this rule are formate/formyl-CoA, which are accepted, and

(methyl)malonyl-CoA, which are not accepted as substrates. The exceptions can be

explained by the small size of format, which allows it to position itself correctly in the









active site, and by the lack of flexibility of (methyl)malonyl-CoA, making it unable to

attain a favorable conformation in the active site. In this regard the large difference in the

transfer rates of CoA to succinate and maleate also indicates a requirement of flexibility

in the CoA acceptor molecule allowing it to make favorable interactions in the active site.

The lack of CoA transfer from propionyl-CoA and acetyl-CoA are consistent with these

substrates not being able to attain a favorable conformation in the active site, presumably

due to their hydrophobic methyl and ethyl groups. Finally, the absence of CoA transfer

to acetate, oxamate, and pyruvate are all indications that without a second negative

charge these molecules are not able to make favorable interactions in the active site.

(A) 0 0
0 0 O O- O 0 0 0
O O
H 0 0 0 0 0 0 0
Formate Oxalate Succinate Maleate Glutarate

(B) 0 0 R
N0- H 0 H2 O- H3 o- 0 0
H o o0 o o

Acetate Propionate Oxamate Pyruvate Malonate (R= H)
Methylmalonate (R = CH3)

Figure 2-8. Structures of free acids and ester parts of CoA esters used for alternative
substrate screening (see Table 2-3). (A) Accepted by FRC as substrates. (B)
Not accepted by FRC as substrates.

Kinetics

As discussed in Chapter 1, kinetic studies are a powerful tool to explore possible

mechanisms of enzyme catalysis. In the case of bisubstrate enzymes yielding two

products, such as FRC, the pattern of lines in a Lineweaver-Burk plot (1/v vs. 1/[S]) of

initial velocity data are a good indication of what type of mechanism the enzyme uses.

Intersecting lines are indicative of a sequential ternaryy complex) mechanism, while









parallel lines suggest Ping-Pong kinetics. In the case of ternary complex mechanisms,

random and ordered ones can be distinguished by product inhibition patterns, and the

order of substrate binding and product release can be deduced in the case of ordered.

Initial Rates

Initial rate measurements in the absence of products were designed so the same

assays would provide data that could be plotted as 1/v versus 1/[formyl-CoA] and 1/v

versus 1/[oxalate] (Figures 2-9 and 2-10). The results were clearly indicative of a

sequential ternaryy complex) mechanism and ruled out Ping-Pong kinetics. The kinetic

constants kcat, Vmax, Km(formyl-CoA), Km(oxalate), and Kia (dissociation constant for the

EA complex) were calculated from the slope and intercept replots (Figures 2-9 and 2-10

inserts), and are summarized in Table 2-4. The Km of formyl-CoA in this study is about

400-fold lower than that reported for the native FRC, while the Km of oxalate is

comparable (Table 1-1). Descriptions of how to calculate the kinetic constants from best-

fit lines of double reciprocal plots and their replots are accessible in Segel's book on

enzyme kinetics.46 Error values were calculated by standard error propagation rules from

the original standard errors in slopes and intercepts of the best-fit straight lines fitted by

using KaleidaGraph (v. 3.5, Synergy Software).

Table 2-4. Kinetic constants of FRC calculated from initial rates data (Figures 2-9 and 2-
10). One unit (U) is defined as one micromole of CoA transferred per minute.
Constant Value
kot 4.3 0.1 s1
Vma 5.5 0.2 U/mg
Km(formyl-CoA) 8.0 + 0.3 IM
Km(oxalate) 3.9 + 0.3 mM
K,, 16 2 uM












04 -

I: 0.8

0/[f-CoA]
01
00 01 Z0.6

25

15 Z_1
10
l) 1/[f-CoA]
050 0
00 0o 1' 0 2


-0.25
-0.25


-0.05-0.1


0.05 0.15


Figure 2-9. Lineweaver-Burk plot of initial rates at three fixed formyl-CoA
concentrations (m 7 aM; o 14 aM; e 65 pM), each at four oxalate
concentrations (5 75 mM). Slope and intercept replots are shown as inserts.
Each data point represents an average of two rate measurements.


04- "

03 0.8


02 [oxalate] g
00 01 02 Z 0.6

35
0.4

SV[oxalate]
1500 01 0
00 01 02 2'.---


-0.05 -0.1


Figure 2-10. Lineweaver-Burk plot of initial rates at four fixed oxalate concentrations (D
5 mM; m 10 mM; o 25 mM; e 75 mM), each at three different oxalate
concentrations (7 65 pM). Slope and intercept replots are shown as inserts.
Each data point represents an average of two rate measurements.










Product Inhibition

The appropriate product inhibitions by format and oxalyl-CoA that would support

or rule out the proposed mechanism (Figure 3-8) were studied. The results are

summarized in Tables 2-5 and 2-6 and discussed below.

Table 2-5. Product inhibition patterns observed for various mechanisms ofbisubstrate
enzymes. The inhibitions observed in this study are shown in bold italic
letters. Substrates and products are A = formyl-CoA, B = oxalate, P =
format, and Q = oxalyl-CoA. The types of inhibitions are C = competitive,
MT = mixed-type (noncompetitive), UC = uncompetitive, and = no
inhibition. Table reproduced from Segel's book on enzyme kinetics.46
Varied substrate
A B
Mechanism Inhibitor Unsaturated Saturated Unsaturated Saturated
with B with B with A with A

Steady-state Ordered P MT UC MT MT
Bi Bi Q C C MT

Steady-state Random P MT MT MT MT
Bi Bi Q MT MT MT MT

Steady-state Ping P MT C C
Pong Q C C MT

Rapid equilibrium P C C
Random Bi Bi Q C -C

Table 2-6. Inhibition constants for format and oxalyl-CoA.*
Inhibit c t V e Slope (Kis) and intercept
Inhibition constant Value ( o
(K,,) composition of K,
K,(formate vs. formyl-CoA) 130 + 20 mM N/A (pure uncompetitive)

Kl = 17 + 1 mM
K,(formate vs. oxalate) 350 + 30 mM K = 3801 4mM
K,, = 380 40 mM
K+ = 150 + 50 M
K,(oxalyl-CoA vs. oxalate) 21 7 M K = 280 + 90 jM
Inhibition constants were calculated as described by Segel46 and error values
were calculated as described above.
Product inhibition by format was measured under three different conditions

(Figures 2-11, 2-12, 2-13). Only one set of conditions was practical for measuring

product inhibition by oxalyl-CoA (Figure 2-14), that is varying oxalate concentration at

fixed, unsaturating formyl-CoA concentration. Making a stock solution with high









enough concentration of formyl-CoA to achieve saturation in the assay mixture is

difficult, and the experiment would waste large amounts of the relatively expensive

substrate without giving much information about the mechanism (Table 2-5). Oxalyl-

CoA could not be studied as a product inhibitor when varying formyl-CoA concentrations

since the initial rate of formation of oxalyl-CoA is being measured, only a relatively large

increase in its concentration can be detected when the assay mixture already contains it.

This is not possible at low formyl-CoA concentrations because it would require using 20-

100% of the substrate, and the reaction would therefore not be at initial velocity.

Inhibition by format

Formate caused mixed-type inhibition when the concentration of formyl-CoA was

varied at a fixed unsaturating oxalate concentration of 350 mM (Figure 2-11), but the

inhibition became uncompetitive with oxalate saturating (1.25 M) (Figure 2-12).

S0.8 -


0.6 -



0.4 -





l/[formyl CoA] (]pM1)

-0.05 0.00 0.05 0.10 0.15 0.20


Figure 2-11. Lineweaver-Burk plot showing product inhibition by format vs. formyl-
CoA at fixed unsaturating oxalate concentration. Formate concentrations
were 0 mM, o 75 mM, and 150 mM. Formyl-CoA concentrations were
5.5 110 [M. The initial concentration of oxalate was 350 mM in all assays.
Each data point represents an average of two rate measurements.



























-0.10


1/[formyl CoA] (pM1)


-0.05 0.00 0.05 0.10


Figure 2-12. Lineweaver-Burk plot showing product inhibition by format vs. formyl-
CoA at fixed saturating oxalate concentration. Formate concentrations were
0 mM, o 20 mM, and 40 mM. Formyl-CoA concentrations were 5.5 -
110 jM. The initial concentration of oxalate was 1.25 M in all assays.
Intercept replot is shown as insert. Each data point represents an average of
two rate measurements.


0.0 0.1 0.2


0.3 0.4
1/[oxalate] (mM 1)


Figure 2-13. Lineweaver-Burk plot showing product inhibition by format vs. oxalate at
fixed unsaturating formyl-CoA concentration. Formate concentrations were
0 mM, o 50 mM, and 100 mM. Oxalate concentrations were 2.5 230
mM. The initial concentration of formyl-CoA was 105 tM in all assays.
Slope and intercept replots are shown as inserts. Each data point represents an
average of two rate measurements.










Formate also caused mixed-type inhibition when the concentration of oxalate was

varied in the presence of fixed unsaturating formyl-CoA concentration (Figure 2-13).

The inhibition was close to being pure competitive, since the slope effect was much

greater than the intercept effect (Table 2-6).

Inhibition by oxalyl-CoA

Oxalyl-CoA functioned as a mixed-type inhibitor when the concentration of oxalate

was varied in the presence of fixed unsaturating formyl-CoA concentration (Figure 2-14).

Relatively high concentrations of oxalyl-CoA were needed to see any inhibition, which

indicates the inhibition will disappear at saturating formyl-CoA concentration, although

this is hard to confirm since very high formyl-CoA concentration is likely needed.


"'S




I0.6 -


[oxalyl-CoA]
015
0 100 200





-0.3 -0.2 -0.1 0 0 0.1 0.2 0.3 0.4
-. l/[oxalate] (mM1)
-0.2


Figure 2-14. Lineweaver-Burk plot showing product inhibition by oxalyl-CoA vs.
oxalate at fixed unsaturating formyl-CoA concentration. Oxalyl-CoA
concentrations were 0 aM, o 90 aM, and m 180 aM. Oxalate concentrations
were 2.5 75 mM. The initial concentration of formyl-CoA was 100 aM in
all assays. Intercept replot is shown as insert. Each data point represents an
average of two rate measurements.

The kinetic measurements of FRC detailed above strongly support an ordered

ternary complex mechanism where formyl-CoA binds first, followed by oxalate, with










format then being released before oxalyl-CoA. Other possible kinetic mechanisms are

effectively ruled out by the results above (Table 2-5). The mechanism is discussed in

more detail in Chapter 3.

Inhibition by Coenzyme A

CoA was of interest as a potential inhibitor, since inhibition by CoA would indicate

binding of it to the active site, and therefore a possibility of seeing it bound in the crystal

structure of FRC incubated with CoA. This would be an example of dead-end inhibition

assuming CoA binds to the free enzyme in the same way formyl-CoA does, but yielding

an enzyme-inhibitor complex that is unable to do catalysis. A mixed-type inhibition

pattern was observed when CoA was used as an inhibitor against oxalate at fixed

unsaturating formyl-CoA concentration is shown in Figure 2-15. This is the expected

pattern when the inhibitor binds only to the free enzyme, and further supports an ordered

Bi Bi mechanism for FRC.62

2.0
S1.5 -


1.0



1/[oxalate] (mM1)

-1.20 -0.70 -0.20 0.30
-0.5 -

-1.0 -

-1.5 -

Figure 2-15. Lineweaver-Burk plot showing inhibition by CoA vs. oxalate at fixed
unsaturating formyl-CoA concentration. CoA concentrations were 35 uM,
o 75 uM, and m 115 gM. Oxalate concentrations were 2.5 50 mM. The
initial concentration of formyl-CoA was 75 uM in all assays. Each data point
represents an average of two rate measurements.















CHAPTER 3
STRUCTURE AND MECHANISM OF FORMYL-COA TRANSFERASE

At the start of this research project, Family III CoA transferases had not been

recognized as a new class of enzymes and protein structures were not available. Crystal

structures of FRC as an apoenzyme and in complex with CoASH became available as a

result of collaboration with a research group at the Karolinska Institute in Stockholm,

Sweden.42,63 Further crystallization experiments yielded a crystal structure of an

acylenzyme intermediate formed when FRC was incubated with oxalyl-CoA.64 These

structures and those of three mutant enzymes are discussed below and how they,

combined with kinetic data from Chapter 2, lend strong support for a novel mechanism of

CoA transfer.

Structure

The crystal structure of FRC reveals a homodimer with a unique assembly of the

subunits. Each monomer consists of a large and a small domain where residues from

both the N- and C-termini of the subunit are part of the large domain. The linkers

between the domains give the subunit an oval shape with a 13 A by 22 A hole in the

middle. In the homodimer the second monomer is threaded through the hole in the first

one and vice versa like two links in a chain (Figure 3-1).





* Structure factors and coordinates have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) under
accession codes 1P5H (FRC), 1P5R (FRC/CoA complex), 1VGQ (D169A mutant), 1VGR (D169E
mutant), 1T3Z (D169S mutant) and 1T4C (FRC/oxalyl-CoA complex).









The interlocked dimer that is observed in the crystal structure of FRC is

inconsistent with previous claims that this enzyme exists as a monomer in solution.1

The oligomeric state of recombinant wild-type FRC was therefore examined using size

exclusion chromatography.

(A) (B)
















Figure 3-1. Monomer and homodimer structures of recombinant formyl-CoA transferase.
(A) One monomer of FRC revealing a large hole in the oval structure. The
protein is shown as molecular ribbons with the N-terminus in blue and gradual
color change through cyan, green, and yellow to the C-terminus shown in red.
(B) The structure of the interlocked FRC homodimer with bound CoA. For
clarity, the protein monomers are colored red and green and represented by
molecular ribbons. The bound CoA molecules are shown as space-filling
models, which are colored using the following scheme: H white; C grey; N
blue; O red; S yellow; P purple.

When 7 utg of FRC were injected on the size exclusion column a broad peak with a

retention time corresponding to a molecular weight of 53.8 kDa resulted. Larger amounts

(30 utg) of FRC, however, yielded a sharp peak matching a mass of 81.0 kDa (Figure 3-

3B). The smaller mass (53.8 kDa) is reasonably consistent with the mass of a monomer

(47.2 kDa) with the deviation between measured and theoretical mass arising from the

open structure of the monomer, and the broadness of the peak probably a result of various










states of unfolding (Figure 3-1A). The larger mass, similarly, is consistent with the

enzyme being a homodimer with the deviation between measured (81.0 kDa) and

theoretical mass (94.4 kDa) arising from the tightly interlocked dimer structure (Figure 3-

1B). This is consistent with the observation that excessive dilution of FRC causes

decreased specific activity of the enzyme (see Chapter 2), and most likely means the

homodimer is disassociating on the size exclusion column when small amounts of sample

are injected.


.............. Monomer (53.8 kDa)
...... ........... .. ,,)



.

KD
0 0.1 0.2 0.3 0.4 0.5


Figure 3-2. Size exclusion chromatography data used to calculate the molecular mass of
FRC. Retention coefficients (KD) of molecular weight standards are shown by
filled circles. Dotted lines show the observed KD for samples of FRC and the
value of logio(MW) calculated from the equation of the best-fit line through
the filled circles. Open circles indicate the placement of FRC samples on the
calibration line. KD is calculated as elutionn volume void volume)/(column
volume void volume).

(A) (B)






bN O o'.o _'o __ __ 2o '

Figure 3-3. Size exclusion HPLC chromatograms of FRC. (A) Broad peak from
smaller amounts of FRC corresponding to 53.8 kDa. (B) Sharp peak from
larger amounts of FRC corresponding to 81.0 kDa.










Active Site

There are two equivalent CoA binding sites in the FRC homodimer, located at the

interface between the large domain of one subunit and the small domain of the other

subunit (Figure 3-1B). The adenine part of CoA is wedged into a thin cleft and buried

from solvent while the ribose, ribose phosphate, and pyrophosphate are solvent-exposed.

The pantetheine chain of CoA is buried in a cleft formed mainly by the large domain.

The small domain participates with a loop composed of residues 258-261 closing off the

cleft where the sulfhydryl group on the pantetheine arm of CoA is bound. In the

apoenzyme structure, this loop adopts an open conformation in one subunit, but has a

closed conformation in the other. In the FRC-CoA complex, this loop is in the closed

conformation in both monomers, leaving insufficient space for an oxalate molecule to

bind in the active site. There is however a suitable binding site for oxalate in the vicinity

of the sulfhydryl group when the loop is in the open conformation (Figure 3-7).






















Figure 3-4. Stereo picture of Coenzyme A in its binding site. Amino acids involved in
hydrogen bonds with CoA are colored according to atom type (C atoms cyan
for FRC and silver for CoA).
Argl04 Axg[04








137 I is1




Iral Vall38



Figure 3-4. Stereo picture of Coenzyme A in its binding site. Amino acids involved in
hydrogen bonds with CoA are colored according to atom type (C atoms cyan
for FRC and silver for CoA).





















































Figure 3-5. CLUSTAL W (1.82) multiple sequence alignment of Family III CoA
transferases. FRC (formyl-CoA transferase from 0. formigenes), YfdW (the
FRC homolog from E. coli), BbsF (succinyl-CoA: (R)-benzylsuccinate CoA
transferase from Thauera aromatica, FldA ((E)-cinnamoyl-CoA: (R)-
phenyllactate CoA transferase from Clostridium sporogenes), CaiB
(butyrobetainyl-CoA: (R)-carnitine CoA transferase from E. coli).









The residues involved in binding of CoA are identified in Figure 3-4. Aspl69 and

Glnl7 are closest to the sulfhydryl end of CoA and Aspl69 is in a position where it could

attack the carbonyl group of a bound CoA ester. Multiple sequence alignment of Family

III CoA transferases is shown in Figure 3-5. YfdW from E. coli is likely a formyl-CoA

transferase since it shares 60% sequence identity with FRC. FRC shared considerably

lower sequence identy with the other transferases, or 26%, 23%, and 20% with BbsF,

FldA, and CaiB respectively (see abbreviations in Figure 3-5 legend). Asp169 was

identified as a residue potentially playing a critical role in catalysis by FRC due to its

position in the active site and because it is conserved in Family III CoA transferases. The

only other residue close to the sulfhydryl group of bound CoA that is fully conserved is

Pro20, which is likely needed for structural purposes creating a critical loop in the

structure. Tyr59 and Tyrl39, which are also close to the active site, are conserved in four

of five sequences shown in Figure 3-5. Tyr59 has been suggested to participate in

stabilizing the oxyanion tetrahedral intermediates that may be formed in the transfer

reaction.42 Tyrl39 makes hydrophobic contacts with the dimethyl group of the

pantetheine chain of bound CoA. Asn96, which contacts CoA, is also conserved in four

of the five sequences.

The relatively low conservation of residues in the active site of Family III CoA

transferases reflects the broad range of substrates that these enzymes use. The location of

the active site, in a cleft between a large domain and a small domain with the sulfhydryl

end of CoA pointing towards the small domain (Figure 3-1B), suggests that the difference

in substrate selectivity between the members of Family III CoA transferases may be

linked to structural differences in the small subunits. This would allow the same overall









protein fold for all members, while creating flexibility in active site structure. As

described above, the small subunit of FRC is part of the active site structure via a flexible

tetraglycine loop consisting of residues 258-261. This moiety is also seen in YfdW, but

not in the other Family III CoA transferases in Figure 3-5, where there is a gap in the

sequence alignment with FRC. This suggests a large difference in the active site

structures, which would explain the difference in natural substrates between these

enzymes.

Point Mutation Studies

Point mutations of FRC were done where Asp 169 was replaced with alanine,

glutamate, and serine to give D169A, D169E, and D169S mutants, respectively. The

mutations were achieved by using mutagenic DNA primers in a polymerase chain

reaction (PCR) with the wild-type FRC in a pET-9a plasmid isolated from the bacterial

expression strain as template. The mutant proteins exhibited the same chromatographic

properties as the wild-type FRC during their purifications. The mutations were

confirmed by DNA sequencing of the mutant plasmids and, later, by X-ray

crystallography (Figure 3-6).

The catalytic activity of the mutated proteins was then assayed using the HPLC-

based assay described in Chapter 2. The sensitivity of the assay allowed up to a 30,000-

fold reduction of CoA transfer rate, relative to wild-type FRC, to be detected. As

expected, the mutations dramatically decreased the transferase activity of the protein.

Surprisingly though, while both the D169E and D169S FRC mutants exhibited no

activity above the detection limit, the specific activity of the D169A mutant was

decreased only 1,300-fold compared to the wild-type FRC. No increase in the rate of

formyl-CoA hydrolysis was detected in these assays, indicating the enzyme had not been










changed to a CoA ester hydrolase by the mutations. Finally, to ensure the loss of CoA-

transferase activity was not due to incorrect folding or quaternary structure of the FRC

mutants, X-ray crystal structures of all three mutants with bound CoA were obtained.

These studies showed that the mutants were correctly folded and formed interlocked

dimers like wild-type FRC. In addition, none of the complexes showed any significant

difference in structure from that observed for the wild-type FRC/CoA structure (Figure 3-

6).


(A)
CoA Oxidised CoA CoA Oxidised CoA
140 6-18 140 16-18

s 169 c(169

Loop 258-261 Loop 258-261
CoA CoA
() Oxidised CoA 10 Oxidised Co
(B) 140 16-18 140 16-18




loop 258-261 Loop 258-261

(C)
COA CoA
S16-18 16-18
169 169




Loop 258-261 '1Loop 258-261


Figure 3-6. Stereo images of active site structures of FRC mutants completed with CoA.
Superposition of the active sites of the CoA complexes of wild-type FRC and
(A) D169A, (B) D169S, (C) D169E. The carbon atoms in the wild-type
FRC/CoA complex are drawn in cyan, and the mutants in purple.

In the complexes involving the D169A and D169S FRC mutants (Figure 3-6A/B),

the sulfhydryl group of CoA appeared to be oxidized, resulting in hydrogen-bonding with

Glul40 rather than Glnl7 and Alal8 as observed in the wild-type FRC/CoA complex.









The reason for this observation only in these two complexes is probably because removal

of the Asp169 carboxyl group leaves the thiol group exposed to oxidation. The

significant loss of transferase activity in these two mutants most likely arises from their

inability to form the key anhydride intermediates (Figure 3-8). The most surprising

observation was the detection of any transferase activity by D169A, which lacks the

active site carboxylate that is apparently critical for normal activity. Since control

experiments ruled out contamination by other CoA transferases, the simplest explanation

for this observation is that the transfer reaction proceeds by a different mechanism,

similar to the one observed for Family II CoA transferases (Figure 1-3B). Thus, oxalate

directly attacks formyl-CoA in a ternary complex to give an oxalyl-formyl anhydride,

which then reacts with bound CoA to yield oxalyl-CoA and format. No rate increase of

formyl-CoA hydrolysis was observed when this substrate was incubated with oxalate and

D169A, which is consistent with the absence of water molecules in the active site of

D169A observed in its crystal structure. Moreover, without wild-type FRC or the mutant,

no oxalyl-CoA formation was detected when formyl-CoA and oxalate were incubated

under the standard assay conditions. This finding is consistent with previous model

studies on the rate of reaction of carboxylic acids with thioesters.65

The conformation of CoA in the active site of the D169E mutant was the most

similar to that observed for the wild-type FRC/CoA complex. However, since the

glutamate side chain is bulkier than that of aspartate the pantotheine group was displaced

slightly (Figure 3-6C), and caused some uncertainty in positioning of the thiol. The lack

of activity in the D169E mutant is probably associated with problems in positioning the









formyl-CoA correctly to permit anhydride formation by reaction of the thioester with the

carboxylate moiety (Figure 3-8).

Mechanism

The mechanism for Family III CoA transferases proposed by Heider was based on

the mechanism of Family II enzymes (Figure 1-3B).25 The enzyme was proposed to

catalyze direct nucleophilic attack of oxalate on formyl-CoA to yield a mixed oxalyl-

formyl anhydride intermediate and free CoA. Addition of CoA at the other end of the

mixed anhydride would then complete the acyl transfer. Notably, no covalent

intermediates would be formed by reaction of the substrates with active site residues in

such a mechanism, the function of the catalyst being primarily to bring the reactants

together in the correct orientation. The crystal structure of wild-type FRC is, however,

not supportive of this mechanism since the position of the side chain of Asp169 appears

to prevent a direct attack of oxalate on formyl-CoA (Figure 3-7).42



Rea 119-142 CA RGs 13q-142 (nA









the surrounding active site. Superimposed to loop 258-26S91 in the closed

Figure 3-7. Stereo picture of the end of the pantetheine chain of CoA and amino acids in
the surrounding active site. Superimposed to loop 258-261 in the closed
conformation is the same loop in the open conformation as seen in the
apoenzyme structure (grey). The two conformations correspond to different
rotamer conformations of Trp48. The cavity formed when the loop is in the
open conformation is shown as a light green cloud. A model of bound
oxalate, in magenta, is included but its orientation is unknown.









0

R1 SCoA R2 -SCoA
R2 0K
0 / 0 0

Asp 0 Asp 0 RI



R2 SCoA
0

R1 SCoA 0 -SCoA

R2 SCoA 0 0

0 --- Asp 0 R2 O
Asp O 0

R1 .0-

Figure 3-8. Proposed mechanism of formyl-CoA transferase from Oxalobacter
formigenes. The box highlights the anhydride intermediate observed by X-ray
crystallography (Figure 3-9). The natural substrates are R1 = H and R2 =
COO Oxygen atoms from the incoming free acid are shown in bold font.

A new hypothesis for the catalytic mechanism of FRC, which includes the direct

involvement of Asp 169, has consequently been proposed (Figure 3-8).42,64 Formyl-CoA

binds to the active site in the initial step and the substrate thioester reacts with the Asp169

side chain to form a covalent formyl-Aspl69 anhydride intermediate and CoA. Oxalate,

entering through the cavity formed when the loop of residues 258-261 is in the open

conformation (Figure 3-7), reacts to generate a new enzyme anhydride intermediate

(oxalyl-Aspl69 anhydride) and format. Subsequent attack of bound CoA on the mixed

anhydride then yields oxalyl-CoA and regenerates the carboxylate moiety of Asp 169.

Since free format is not produced until after oxalate is bound, kinetic plots cannot

assume the form observed for classical Ping-Pong mechanisms like in the case of Family

I CoA transferases.









The proposed mechanism of Family III enzymes predicts incorporation of 80 into

the main catalytic residue (Figure 3-8) as for Family I enzymes (Figure 1-3A) if an 80-

labeled CoA acceptor would be used. This has not yet been verified for any Family III

CoA transferase. An important difference between the proposed mechanism of Family

III CoA transferases and the Family I CoA transferase mechanism is the absence of

covalent enzyme-CoA intermediates in the former. Family I CoA transferases are

completely inactivated when incubated with hydroxylamine in the presence of one of

their CoA ester substrates. The inactivation is a result of the reaction of hydroxamic

acids, formed from the hydroxylamine and the thioester substrate, with the CoA ester of

the main catalytic residue (a glutamic acid) (Figure 1-3A).34 Studies on two Family III

enzymes support the absence of covalent enzyme-CoA intermediates in the mechanism of

Family III CoA transferases. Neither BbsF (succinyl-CoA: (R)-benzylsuccinate CoA

transferase) nor FldA ((E)-cinnamoyl-CoA: (R)-phenyllactate CoA transferase) are

inactivated by hydroxylamine in tests with and without the CoA ester substrate.27'29

NaBH4 has also been shown to inactivate Family I enzymes by reacting with the

covalent enzyme-CoA intermediate reducing the glutamic acid residue to an alcohol.33'35

Family III CoA transferases are at least partially inactivated by NaBH4, although

inactivation requires higher concentration of NaBH4 than for Family I enzymes. The

activity of BbsF decreased by 75% after incubation with 0.25 mM benzylsuccinyl-CoA

and 1 mM NaBH4 at pH 7.5 for 10 minutes27 and the activity of FldA decreased by 50%

after incubation with 49 pM cinnamoyl-CoA and 10 mM NaBH4 at pH 7 for 15

minutes.29









The simplest interpretation of these observations is that there is no covalent

enzyme-CoA intermediate present during catalytic turnover of Family III CoA transferase

enzymes, which would explain them not being inactivated by hydroxylamine.

Furthermore, the relatively slow inactivation by NaBH4 is then due to the reduction of a

covalent enzyme-substrate anhydride intermediate that is less reactive and/or not as

accessible as the covalent enzyme-CoA thioester intermediates in the Family I

mechanism. This may be possible to confirm using tritiated sodium borohydride as has

been done for Family I enzymes.66 NaBH4 has been used to reduce mixed anhydrides of

carboxylic acids and carbonic acids,67 and cyclic carboxylic acid anhydrides.68




CoA CoA

Oxalyl aspartic Oxalyl aspartic
anhydride anbydride
Gin B 17 Gin B17




B140 140
loop A258-A261 loop A25S-A261

Figure 3-9. Stereo picture showing the interactions of the oxalyl-aspartyl anhydride with
residues in the FRC dimer. The letter designation (A or B) in the numbering
scheme indicates the FRC monomer in which the residue is located.

Obtaining protein crystals of FRC with bound formyl-CoA or oxalyl-CoA proved

elusive due to the liability of the thioesters. Surprisingly, after extensive screening

studies, the product of reaction between FRC and oxalyl-CoA, i.e. the putative oxalyl-

Asp169 anhydride intermediate (Figures 3-8 and 3-9), was crystallized by Stefano

Ricagno, a graduate student in the laboratory of Dr. Ylva Lindqvist, the crystallography

collaborator.64 This structure lends very strong support to the participation of mixed






46


anhydrides of Asp169 in the catalytic mechanism of FRC, and along with the kinetic data

presented in Chapter 2 strongly supports the proposed ordered Bi Bi mechanism shown in

Figure 3-8.














CHAPTER 4
EXPERIMENTAL

All materials were of the highest purity available and, unless stated otherwise,

obtained from Fisher Scientific International, Inc. (Hampton, NH) or Sigma-Aldrich

Corp. (St. Louis, MO). Protein concentrations were determined by the Lowry method as

modified by Hartree69 using bovine serum albumin as a standard. DNA sequencing was

performed by the DNA Sequencing Core of the Interdisciplinary Center for

Biothechnology Research at the University of Florida. The BL21(DE3) Escherichia coli

expression strain transformed with pET-9a plasmid carrying the gene for wild-type FRC

was supplied by Dr. Harmeet Sidhu (Ixion Biotechnology, Inc., Alachua, FL). Mutagenic

PCR (polymerase chain reaction) primers were obtained from GenoMechanix LLC

(Gainesville, FL) (D169A and D169E) and Integrated DNA Technologies, Inc.

(Coralville, IA) (D169S).

Expression and Purification of FRC

Wild-type formyl-CoA transferase (FRC) from Oxalobacterformigenes was

overexpressed in BL21(DE3) Escherichia coli and purified by anion exchange and

affinity chromatography. The enzyme was expressed by growing an overnight culture

(100 mL) in Luria-Bertani broth containing 30 [tg/mL kanamycin (LBK) at 370C and

200-250 rpm, which was then used to inoculate 4-6 L of fresh LBK. FRC expression was

induced by adding IPTG (isopropyl-,/-D-thiogalactopyranoside) when the optical density

of the culture, grown at 370C and 200 rpm, reached 0.6-0.8 at 600 nm (4 mL of 0.1 M

IPTG added per liter of culture). The cells were harvested 2.5-4.0 hours after induction









by centrifugation at 40C. The cell pellets were resuspended in 50-150* mL lysis buffer

(100 mM potassium phosphate, 1 mM dithiothreitol (DTT), pH 7.2) and lysed by passing

two times through a French press or by sonication (ten 10 second pulses with 30 second

intervals). After centrifuging, the lysate supernatant was loaded on a 120 mL DEAE Fast

Flow anion exchange column equilibrated with buffer A (25 mM sodium phosphate, 1

mM DTT, pH 6.2) and FRC eluted by stepping to 35% buffer B (25 mM sodium

phosphate, 1.0 M NaC1, 1 mM DTT, pH 6.2) at 5 mL/min. The FRC-containing fractions

were loaded on a 20 mL Blue FF (Blue SepharoseTM 6 Fast Flow) affinity column

equilibrated with buffer A. The column was then washed with 50% buffer B and FRC

finally eluted with buffer C (25 mM glycine, 1 mM DTT, 20% isopropanol, pH 9.0) at 4

mL/min. The FRC-containing fractions from the affinity column were immediately

buffer-exchanged by passing through a 135 mL G-25 desalting column equilibrated with

buffer A at 10 mL/min. The protein solution was then injected on a 60 mL Q-Sepharose

high performance anion exchange (QHP) column equilibrated with buffer A and the FRC

eluted at 5 mL/min by stepping to 20% buffer B for 1-2 column volumes followed by a

linear gradient to 35%B over 6-7 column volumes, with FRC eluting close to the center

of the linear gradient. Glycerol was added to achieve a concentration of 10% to stabilize

the protein since it has a tendency to precipitate. The resulting protein solution was

stored at -800C. All purification steps were run at ambient temperature and the FRC

fractions stored on ice between purification steps. The purification was verified by SDS-

PAGE with Coomassie Blue staining and activity measurements of the fully purified

enzyme. See Appendix A for typical FPLC chromatograms from each purification step.


*Smaller volumes used when lysing with French press. Larger volumes used when lysing by sonication.









The pooled FRC-containing fractions after the first three chromatography steps

have a tendency to form a protein precipitate, resulting in loss of CoA transferase

activity. This loss is minimized by using 1 mM dithiothreitol (DTT) in all buffers and

storing the collected fractions on ice until the next purification step. For best yields, the

first three steps should be performed within 12 hours. The fully purified FRC eluted off

the QHP column also has a slight tendency to precipitate, but addition of glycerol to a

concentration of 10% stabilizes the protein so no precipitate is visible after freezing and

thawing. The Blue FF affinity column resin has a lifetime of about one year when

storage instructions are followed and using a column that has started to degrade

dramatically decreases yields.

Expression of Selenomethionine Derivative of Wild-Type FRC

The selenomethionine derivative of wild-type FRC (SeMet FRC) was prepared

using literature procedures for expression of SeMet proteins in nonauxotrophic strains of

E. coli.51 The bacteria were grown in M9 medium (2 mM MgSO4, 0.1 mM CaCl2, 48

mM Na2HPO4, 22 mM KH2PO4, 9 mM NaC1, 19 mM NH4C1, 4 g/L glucose) at 370C and

200 rpm until the optical density of the culture at 600 nm reached 0.6-0.8. Methionine

biosynthesis by the bacteria was then downregulated by addition of lysine (100 mg/L),

threonine (100 mg/L), phenylalanine (100 mg/L), leucine (50 mg/L), isoleucine (50

mg/L), valine (50 mg/L), and proline (50 mg/L). Selenomethionine (50 mg/L) was then

added to the culture, which was incubated for 15 minutes before inducing with IPTG as

described for wild-type FRC above. The cells were harvested 4-5 hours after induction

and the SeMet FRC purified as described for wild-type FRC.









Site-Directed Mutagenesis

Mutagenic primers were designed using Gene Runner v. 3.05 (Hastings Software,

Inc.). The pET-9a plasmid with the FRC gene insert was isolated from BL21(DE3) E.

coli with the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI)

and used as a template for PCR with the mutagenic primers using the QuikChange Site-

Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The desired mutations were

verified by DNA sequencing of the FRC gene inserts of the mutated pET-9a plasmids

isolated from transformed XL-1 or XL-10 Gold supercompetent cells (Stratagene, La

Jolla, CA). BL21(DE3) competent cells (Novagen, Madison, WI) were then finally

transformed with the mutated plasmids and the mutated FRC proteins expressed and

purified as described for wild-type FRC above.

Synthesis of CoA Esters

Formyl-CoA

Formylthiophenyl ester was made by formylating thiophenol53 using a formylating

reagent made from acetic anhydride and formic acid.54 Formic acid (6.9 g; 150 mmols;

5.8 mL) was added dropwise to stirred acetic anhydride (7.7 g; 75 mmols; 7.1 mL) and

the resulting mixture stirred at 45 C for 2.5 hours. Pyridine (59 mg; 0.75 mmols; 61 |tL)

was then added, immediately followed by thiophenol (5.5 g; 50 mmols; 5.1 mL), and the

reaction mixture stirred at room temperature for 24 hours and then stored at 7 C

overnight after purging with dry nitrogen gas. Thin layer chromatography (TLC) (1:1

chloroform:hexanes) showed the product at Rf= 0.53 and no reactant (Rf= 0.72) with a

faint spot at Rf= 0. The remaining acids and anhydrides were removed from the reaction

mixture by vacuum distillation (40 500C/20 mmHg) and collected in a cold-finger trap

immersed in liquid nitrogen. A water aspirator was used, connected via a drying tube to









the reaction flask. When the volume had decreased to 6-8 mL the product was washed

with an equal amount of deionized water and then dried over anhydrous magnesium

sulfate. TLC, after washing with water, indicated minor decomposition of the product.

The product was finally vacuum distilled (115-117 C/23 mmHg), yielding 3.1 g or 45 %

of pure product. The literature values are 101 C/15 mmHg and 87 % yield. 1H-NMR

(CDC13, 300 MHz) 6 10.22 (s, 1), 6 7.46 (m, 5). 13C-NMR (CDC13, 75.5 MHz) 6 190.0,

134.1, 129.9, 129.5, 126.0. See NMR spectra in Appendix B.

Formyl-CoA was prepared by a method based on the procedure by Sly and

Stadtman.52 Sodium salt of CoA (80 mg, 96 [tmols CoA) was dissolved in 2-8 mL of ice-

cold deionized water and the pH adjusted to 7.0 with 0.1 M KOH or NaOH while cooling

on ice. Formylthiophenyl ester (80 mg, 580 tmols) was dissolved in ice-cold dry ether

(1-2 mL) and added quickly to the cold CoA solution while stirring, resulting in pH

decrease to 6.6. The pH was adjusted to 7.5-8.0 with 1 M KHCO3 or NaHCO3 at pH 8.0

(0.25-1.0 mL) and the reaction mixture stirred on ice for 2-3 minutes. The pH was then

carefully adjusted to 3.0 with 0.1 M HC1 under rapid stirring, and the solution washed

three times with two volumes of ice-cold ether. The pH was finally adjusted to 5.5 with

0.1 M KOH or NaOH and stored at -800C. Typical yields were about 70-90%.

LC/(+/-)ESI-MS (Flow Injection Analysis) calculated for C22H36N7017P3S: 795.5; found:

795.1 (+)ESI-MS and 795.6 (-)ESI-MS.

Oxalyl-CoA

Thiocresol oxalate (25 mg, 0.13 mmol) dissolved in ice-cold anhydrous ether (5-10

mL) was added slowly to an ice-cold solution of CoA (sodium salt) (25 mg, 0.031 mmol)

in water (10 mL) at pH 7.5 (adjusted with 0.1 M NaHCO3). The reaction mixture was

stirred on ice for about 20 min before removing the aqueous layer and acidifying it









carefully to pH 3.5 with 0.1- 1.0 M HC1 followed by washing with two 12 mL portions of

ice-cold ether. When complete removal of thiocresol oxalate was desired the aqueous

phase was washed eight times with ether adjusting the pH of the aqueous phase to 3.0

between washes. The resulting solution was stored at -80 OC. Typical yields were 80-

100%. C-18 HPLC/UV(260 nm)/(+)ESI-MS calculated for C23H36N7019P3S: 839.5;

found: 839.4.

Analysis of CoA Esters

Analysis of the CoA esters was based on literature procedures57 using C-18

reversed-phase HPLC (Dynamax Microsorb 60-8 C18, 250 x 4.6 mm) with a single-

wavelength detector at 260 nm. The column was equilibrated with 86% buffer A (25 mM

NaOAc, pH 4.5) and 14% buffer B (20 mM NaOAc, pH 4.5, 20% CH3CN) running at 1.0

mL/min. Immediately after injection a 12 minute gradient to 34% buffer B was started

followed by a step to 100% buffer B for 2 minutes before returning to 14 % buffer B.

Oxalyl-CoA eluted at 6.5 minutes, free CoASH at 11.5 minutes, and formyl-CoA at 12.5

minutes. The CoA esters were quantitated by integration of their peaks in the HPLC

chromatograms using free CoASH as a quantitative standard. The validity of this method

was confirmed for formyl-CoA by independently measuring its concentration in solution

using the hydroxylamine method,58-60, and for oxalyl-CoA by measuring the

concentration of oxalate in an oxalyl-CoA solution before and after complete hydrolysis

by base using an oxalate detection kit (Sigma-Aldrich Corp., St. Louis, MO).

Purification of CoA Esters

The CoA esters were purified using a preparative C-18 reversed-phase column

(Dynamax 60A C18, 250 x 21.4 mm). The column was equilibrated with 88% mobile

phase A (10 mM sodium phosphate at pH 5.0) and 12 % mobile phase B (mobile phase A









with 20% acetonitrile) at 10 mL/min. Two minutes after injecting the sample the fraction

of mobile phase B was increased linearly to 38% over 17 minutes. The absorbance of the

eluent at 260 nm was monitored and fractions collected manually on ice. Oxalyl-CoA

eluted at about 5 min, CoA at 12 min, and formyl-CoA at 13 min. The fractions were

analyzed as described above and lyophilized in small (1-2 mL) aliquots.*

Enzymatic Assay

The recombinant wild-type FRC was assayed by measuring the initial rate of

oxalyl-CoA formation by HPLC analysis of quenched aliquots. The assay mixture

contained 60 mM potassium phosphate (pH 6.7), FRC (90 ng, 9.5 nM), and variable

amounts of substrates and inhibitors (if desired) in a total volume of 200 pL. The

reaction was started by addition of formyl-CoA after incubating the other components at

30 C for about 30 seconds. Aliquots of the reaction mixture (90 [tL) were typically

taken after 60 s and 90 s and quenched with 10% acetic acid** (10 [tL) before quantitating

oxalyl-CoA by reversed-phase HPLC using a shorter version of the analytical procedure

described above that separates only oxalate and oxalyl-CoA from the rest of the mixture

components.64 No formation of oxalyl-CoA was detected in control experiments when

the enzyme or either substrate was omitted, or when FRC denatured by incubation in

boiling water was used. The limit of detection of the assay as described is about 0.05 [tM

of oxalyl-CoA when 75 [tL of quenched assay mixture is injected on HPLC column.

The specific activities of the D169A, D169E, and D169S FRC mutants were

assayed using an identical procedure except that reaction mixtures were incubated for up


*Small aliquots minimized hydrolysis of the CoA esters during freeze-drying.

SThe pH of the quenched aliquots should be 3-4, which is ideal for CoA ester stability. Oxalyl-CoA
concentration in quenched samples remains unchanged for several hours when stored on ice.









to 60 minutes prior to quenching. In addition, because of the much lower activity of the

FRC mutants, the amount of enzyme in each assay was increased to 2 [tg, and the initial

concentrations of oxalate and formyl-CoA were 100 mM and 200 PM, respectively.

Alternative substrates (Table 2-3) were screened using the assay described above,

replacing the natural substrates with the substrates being tested. CoA ester

concentrations were in the range of 80-200 [aM and free acid concentrations were 62.5-

125 mM in these experiments.

Equilibrium Constant Determination

Wild-type FRC (18 atg) was incubated at 220C with 73 [aM formyl-CoA, 50 [aM

oxalate, 13 [aM format, and 13 [aM free CoA (introduced by formyl-CoA stock solution)

in 60 mM potassium phosphate buffer at pH 6.7 (200 atL total volume). Aliquots (45 pL)

were withdrawn after 10, 27, and 52 minutes, and quenched with 10% acetic acid (5 aL).

The concentrations of oxalyl-CoA, free CoA (and therefore formate, and formyl-CoA in

each sample was measured by HPLC as described above. Equilibrium concentrations

were reached after 27 minutes, giving Keq = 32 + 3.

Size Exclusion Chromatography (SEC)

A BIOSEP SEC-S2000 column (300 x 7.8 mm with 75 x 7.8 mm guard column)

(Phenomenex, Torrance, CA) was equilibrated with 100 mM potassium phosphate at pH

6.6 running at 1.0 mL/min. The column was calibrated using lysozyme (14.4 kDa),

carbonic anhydrase (29.0 kDa), peroxidase (44.0 kDa), bovine serum albumin (66.0

kDa), alcohol dehydrogenase (150 kDa), and P-amylase (200 kDa). The void volume of

the column was measured by injecting blue dextran. Samples of FRC (7 ag and 30 pg)

were injected on the column and the molecular weights (53.8 kDa and 81.0 kDa)

calculated from the retention times.














CHAPTER 5
SUMMARY

This research project was originally aimed at determining whether the mechanism

of FRC was a Ping-Pong mechanism, used by almost all known CoA transferases known

at the time. However, upon discovering that FRC belongs to a new class of CoA

transferase enzymes, apparently using a novel mechanism of CoA transfer, the focus of

the project turned to deciphering that mechanism. The unique crystal structure of FRC

and sequence alignments of Family III CoA transferase enzymes identified a putative

main catalytic residue, which was confirmed by its mutation and the resulting loss of

activity, thus refuting a mechanism like the one used by Family II CoA transferases as

proposed previously.25 Following synthesis of substrates and development of an

enzymatic assay, steady-state kinetic studies and product inhibition patterns led to the

proposal of a novel mechanism of CoA transfer, which includes covalent enzyme-

substrate anhydride intermediates. One of these putative intermediates was observed by

X-ray crystallography of FRC crystals grown in the presence of oxalyl-CoA, providing

further evidence for the proposed mechanism. Since the main catalytic residue is

conserved in known Family III CoA-transferases, the catalytic mechanism of formyl-

CoA transferase is almost certainly employed by all other members of this enzyme

family.

Although no direct evidence for the proposed covalent enzyme-substrate anhydride

intermediates exist for Family III CoA transferases the crystal structure data presented

herein are all supportive of such intermediates. An 0O-exchange experiment using 10-









oxalate with formyl-CoA or 180-formate with oxalyl-CoA should provide definitive

evidence for these intermediates (Figure 3-8) as it has for Family I CoA transferases.32

So far, no convenient proteolytic conditions, which are necessary for such experiments,

have been found for FRC.

The possibility of a covalent enzyme-CoA thioester in the mechanism of FRC, as

observed for Family I CoA transferases (Figure 1-3A), can be tested by using the

methods of Hersh and Jencks.33 After incubation of the enzyme with a CoA ester

substrate and subsequent removal of small molecules by size exclusion filtration the

presence of free CoA can be assayed after allowing the putative enzyme-CoA

intermediate to hydrolyze. Incubation of the enzyme with free CoA serves as a control

reaction, since no CoA should be detected in the protein fraction after the size exclusion

filtration.

Some other questions remain regarding the proposed mechanism, such as the

timing of the mixed anhydride formation, which could take place before or after oxalate

binding, although the observation of the aspartyl-oxalyl anhydride intermediate in the

crystal structure would suggest this happens in the absence of the free acid substrate,

since there was no oxalate or format present in the solution the crystals were grown in.

It is not known whether CoA stays bound after the mixed anhydride formation or if it

diffuses into solution. In theory, at least the pantotheine moiety may have to move out of

the active site to allow the incoming free acid to attack the mixed anhydride, unless the

attack comes from the other side of the anhydride intermediate. One of the biggest

questions concerns the substrate selectivity of FRC. The features of the active site

responsible for the high selectivity towards format, oxalate, and succinate, while acetate,









oxamate, and pyruvate are not accepted as substrates, are not yet known. Perhaps

computational modeling can provide some answers to these questions, identifying

favorable or unfavorable interactions between active site residues and the bound

molecules. More site-directed mutagenesis studies aimed at residues in the active site

known or suspected of interacting with the bound substrates, such as Glnl7, Tyr59, and

Tyrl39, would also undoubtedly be beneficial to the understanding of the mechanism.

Another issue is the discovery of the yfdW gene in E. coli and the expression of its

protein product, whose crystal structure is the same as FRC's. E. coli has never been

shown to degrade oxalate, although the presence of this gene and the orthologous gene of

oxalyl-CoA decarboxylase from 0. formigenes would indicate that it is capable, given

that these proteins have the same function as in 0. formigenes. An interesting experiment

would be to examine whether E. coli can be induced to express these genes and become

an active oxalate degrader. Furthermore, comparing the activities and substrate

specificities of the enzymes from these two sources would be of value.

Given that FRC and oxalyl-CoA decarboxylase (OXC) combined constitute up to

20% of the total protein content of 0. formigenes,11'13 and the important function they

serve in the cell, it is not unreasonable to suggest that these enzymes may interact

somehow, perhaps creating oxalate-degrading complexes inside the cell. This would be

beneficial to the organism, especially since it would minimize spontaneous hydrolysis of

the labile formyl-CoA, which would effectively stop the catalytic cycle of oxalate

breakdown. The fact that the adenine part of CoA is firmly bound to FRC while the rest

of the molecule is solvent accessible could mean that the pantetheine arm of CoA

esterified with oxalate can swing out of the active site of FRC directly into the active site






58


of OXC and return to FRC as a formyl ester after decarboxylation. This hypothesis could

be tested by fluorescence labeling and/or immunohistochemistry methods.

Finally, understanding the folding mechanism of the remarkable interlocked dimer

of FRC could be a large contribution to the field of protein folding, and collaboration in

that regard is currently under way.














APPENDIX A
PROTEIN PURIFICATION CHROMATOGRAMS

The following figures show typical chromatograms of the four purification steps

used to purify the wild-type, selenomethionine, and mutated formyl-CoA transferases.


Figure A-1. FPLC chromatogram of the DEAE anion exchange purification step. Blue
Absorbance at 280 nm (0-3000 mAu*). Red = Conductivity (scale 0-75
mS/cm). Black = Percent concentration of buffer B (scale 0-100%). The
arrow points to the FRC containing peak.











mAu = milli absorbance unit




























Figure A-2. FPLC chromatogram of the BlueFF affinity purification step. Blue =
Absorbance at 280 nm (0-1700 mAu). Red = Conductivity (scale 0-45 mS/cm). Black
Percent concentration of buffer B (scale 0-50%). The arrow points to the FRC peak.







60 0











Figure A-3. FPLC chromatogram of the buffer exchange purification step. Blue =
Absorbance at 280 nm (0-750 mAu). Red = Conductivity (scale 0-22
mS/cm).


y-:































Figure A-4. FPLC chromatogram of the QHP anion exchange purification step. Blue
Absorbance at 280 nm (0-120 mAu). Red = Conductivity (scale 0-75
mS/cm). Black = Percent concentration of buffer B (scale 0-100%). The
arrow points to the FRC peak.




















APPENDIX B
NMR SPECTRA OF FORMYLTHIOPHENYL ESTER


The following figures in show the 1H-NMR and 13C-NMR spectra of


formylthiophenyl ester, the formylating reagent used to prepare formyl-CoA. See


experimental section (Chapter 4) for chemical shifts.


I-


. ___ 'B
a s


7 6 5 4 3 1 ppm


Figure B-1. 1H-NMR spectrum of formylthiophenyl ester.


_ ,1__,_

10
lr^
iatO













































120 lI bU


Figure B-2. 13C-NMR spectrum of formylthiophenyl ester.





































190 180 170 160 150 140 130 ppm


Figure B-3. Close-up on relevant peaks of Figure A-2.


4V -u 0pPl


?MD
















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BIOGRAPHICAL SKETCH


Stefan J6nsson was born in Reykjavik, Iceland, in 1972. He holds a B.Sc. degree

in biochemistry from the University of Iceland and an M.Sc. degree in chemistry from the

same school. After working one year as a research scientist for deCODE Genetics, Inc in

Reykjavik he started graduate studies in the Chemistry Department of the University of

Florida in August 1999, where he joined Dr. Nigel G. J. Richards' research group in May

2000.