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Proton transfer in catalysis by the carbonic anhydrases

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Proton transfer in catalysis by the carbonic anhydrases
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Earnhardt, J. Nicole, 1970-
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PROTON TRANSFER IN CATALYSIS BY THE CARBONIC ANHYDRASES













By

J. NICOLE EARNHARDT

















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 1998














ACKNOWLEDGMENTS


I would foremost like to extend my deepest appreciation to my mentor, Dr. David Silverman. Dr. Silverman has provided me with a productive, motivational and well-funded environment to conduct my research. His tutelage has given me the self-confidence to ensure every project I pursue is successful. I am also indebted to Dr. C. K. Tu for his countless hours of time and patience in teaching me instrumentation operations and his unselfish assistance with data analysis, experiments, troubleshooting and brainstorming. Dr. Minzhang Qian was invaluable to my research; she was responsible for generating most of the mutant forms of carbonic anhydrases, which I used for my doctoral research. I want to thank Dr. Philip Laipis for being co-chairman of my graduate committee and for his significant contributions to my work and development as a scientist. I would also like to thank past and present technicians of the Silverman and Laipis laboratories, including Bret Schipper and Nina Wadhwa, as well as our collaborators from the laboratories of Richard Tashian and Ronald Viola. Also, I thank my friends in the Silverman lab, who have not only been helpful and provided sound advice but have also been a real pleasure to work with every day. I would like to thank Dr. Brian Cain for providing assistance during my change in laboratories in my third year. I would like to thank Dr. Harry Nick for taking a specific interest in my success as a graduate student. He has always treated me as one of his own students since I came to the University of Florida. I am looking forward to working with him during my postdoctoral studies.

11








Most importantly, I would like to thank the people closest to my heart. My father, brother, and grandma are truly my best friends for life. I know I could never fail in anything I pursue with my family supporting me. To Chris: a simple thank you cannot suffice for someone who guided me through the roughest times of my life and graduate career. He has made my fife everything that it should be and even more. Knowing Chris and his dear family has made me become a better person every day.














TABLE OF CONTENTS


page

ACKNOWLEDGMENTS...........................................11i

LIST OF TABLES............................................... vii

LIST OF FIGURES.............................................. viii

ABSTRACT ...................................................... x

CHAPTERS

1. INTRODUCTION........................................... I
The Mammalian Carbonic Anhydrases .............................1I
Physiological Function...................................1I
Structure ............................................ 3
Inhibition ............................................ 7
Overview of the Catalysis....................................... 8
Interconversion of CO2 and HCO3;.......................... 9
Proton Transfer .. . .. . 1
Bronsted Analysis....................................... 14

2. THE CATALYTIC PROPERTIES OF MURIh1E CARBONIC
ANHYDRASE VII ............................................ 18
Introduction................................................. 18
Materials and Methods......................................... 20
Expression and Purification of a Recombinant Murine CA VII cDNA 20 Subcloning and Site-Directed Mutagenesis of CA VII .............23
Enzyme Purity......................................... 26
Stopped-Flow Spectrophotometry ...........................26
'0 Exchange .. . . .27
Inhibition ........................................... 29
Hydrolysis of 4-Nitrophenyl Acetate ........................ 31
Results .................................................. 31
Recombinant Murine CA VII ............................. 31


iv









Catalytic Activity...................................... 32
Discussion................................................ 40
Comparison of Isozymes of Carbonic Anhydrase................ 40
Proton Transfer in CA VII ............................... 42
Assignment of pK, Values ............................... 46
Inhibition ........................................... 49
Conclusions ......................................... 49

3. INTRAMOLECULAR PROTON TRANSFER FROM MULTIPLE SITES IN
CATALYSIS BY MURINE CARBONIC ANHYDRASE V .............51
Introduction . . . ..51
Materials and Methods ....................................... 53
Site-Specific Mutagenesis................................ 53
Expression and Purification ......................... 55
Stopped-flow Spectrophotometry .......................... 56
"0O Exchange ........................................ 56
Results .................................................. 56
Discussion................................................ 66

4. CATALYSIS BY MURINE CARBONIC ANHYDRASE V IS ENHANCED
BY EXTERNAL PROTON DONORS............................ 75
Introduction . .. .. ..75
Materials and Methods................................... .... 77
Site-Specific Mutagenesis, Protein Expression and Purification ......77 "0O Exchange ........................................ 78
Results .................................................. 78
Method I: Saturation Effect of Buffers on RH~d[EI ...............78
Method 2: pH Dependence of RH2d/[E] ..........................81
pK. of the Donors and Acceptors Listed in Table 4-1 .............86
Determination of kB Values of Table 4-1 by Method I.............88
Solvent Hydrogen Isotope Effects.......................... 89
Discussion................................................ 89
Choice of Mutant and Buffers ............................. 89
Enhancement of Catalysis................................ 90
Bronsted Analysis ..................................... 91
Marcus Rate Theory ................................... 94
Conclusions ......................................... 97

5. DISCUSSION, CONCLUSIONS AND FUTURE DIRECTIONS .........99
Isozyme VII............................................... 99
Isozyme V ............................. ................. 102
Buffers in Catalysis......................................... 103
Conclusions.............................................. 104


V









Future W ork ................................................. 105

RE FERE N C E S ..................................................... 108

BIOGRAPHICAL SKETCH .............. ............................ 114













































vi















LIST OF TABLES


Table page

1-1 Cellular Location and Predominant Tissue Distribution of the Mammalian Carbonic Anhydrases ............................................ 2

1-2 Maximal Values of the Steady-State Constants with Values of Apparent pKa for CO2 Hydration Catalyzed by the Mammalian Carbonic Anhydrases ... 10

2-1 Inhibition Constants K, (Nanomolar) for Isozymes of Carbonic Anhydrase Determined by "S0 Exchange ....................................... 30

2-2 Catalysis by Carbonic Anhydrase VII: Maximal Values of Steady-State Constants with Values of Apparent pKa ............................... 34
3-1 Maximal Values ofkca/Km and kat for CO2 Hydration and kB with pKa Values Obtained from their pH Profiles for Wild-type and Mutants of Murine
Carbonic Anhydrase V ....................................... 57

3-2 Catalysis Enhanced by Proton Donors from Solution in Mutants of MCA V: Values of RH,2/[E], the Rate of Release of '80-labeled Water from the
A ctive S ite .. ......... ......... ........................ .. ..... 65

4-1 Constants of Equations 4-1 and 4-2 for the Enhancement by Buffers of Catalysis by Y64A/F65A M CA V ................................... 80

4-2 Maximal Values of kB with pKa Obtained from their pH Profiles for the Y64A/F65A Mutant of Murine Carbonic Anhydrase V, in the Absence and
Presence of Buffer: M ethod 2 ...................................... 85

4-3 Marcus Theory Parameters for Proton Transfer in Isozymes of Carbonic A nhydrase .................................................... 96





vii















LIST OF FIGURES


Figure pae

1-1 A ribbon model of human carbonic anhydrase II determined from the crystal structure ................................................. 4

1-2 Residues near the active site of human carbonic anhydrase II ............... 6

2-1 The sequence of MCA VII cDNA and derived amino acids ............... 21

2-2 The pH dependence of ku/Km for hydration of CO2 and dehydration of HCO3 catalyzed by rM CA VII at 25 C .................................... 33

2-3 The turnover number k, for hydration of CO2 and dehydration of HCO3catalyzed by rMCA VII and hydration of CO2 catalyzed by rMCA VII H64A
obtained by stopped-flow spectrophotometry at 25 C ................... 36

2-4 Variation with pH of RH2o/[E], the proton-transfer dependent rate constant for the release from the enzyme of "O-labeled water, catalyzed by rMCA VII
and the H64A mutant of rMCA VII ............ ... ....... 38

2-5 The variation with pH of RH2/[E] catalyzed by full-length rMCA VII, the H64A mutant of full-length rMCA VII, the truncated MCA VIIb, and a
H64A mutant of the truncated MCA VIIb ............................ 39

2-6 The pH dependence of k.jK. for the hydrolysis of 4-nitrophenyl acetate catalyzed by rM CA VII .......................................... 41

3-1 The location of ionizable residues near the active site cavity of murine carbonic anhydrase V from the crystal structure ............................... 54

3-2 The pH dependence of ku.Km for hydration of CO2 determined by 0 exchange catalyzed by wild-type MCA V; K91 AY131A MCA V; and
Y64A/K91A/Y131A MCA V at 25 0C ............................... 58



viii








3-3 The pH dependence of kc., for hydration of CO2 determined by stopped-flow
spectrophotometry catalyzed by wild-type; K91A/Y 131A MCA V; and
Y64A/K91A/YI31AMCA V at25 0C. ........... ............... 60

3-4 The pH dependence of Rmo/[E], the rate constant for release of O-labeled
water from the enzyme, catalyzed by wild-type MCA V and the mutant
K91A/YI31AMCA V at25 oC ............................... 62

3-5 The dependence of Rmo/[E] and R/[E] as a function of the concentration of
imidazole at pH 6.3 and 25 C ................ ................. 64

4-1 The dependence of RH20/[E] and R,/[E] as a function of the concentration of
protonated 3,5-dimethyl pyridine at pH 6.3 and 25 C.................... 79

4-2 Variation with pH of RH20/[E], the proton-transfer dependent rate constant
for the release from the enzyme of "O80-labeled water, catalyzed by MCA V
Y64A/F65A in the absence of buffer and in the presence of 100 mM
3,5-dimethyl pyridine and 100 mM imidazole at 25 C .................. 82

4-3 The difference in RH2mo/[E], the proton-transfer dependent rate constant for the
release from the enzyme of "O-labeled water, between MCA V Y64A/F65A
in the absence and presence of 100 mM 3,5-dimethyl pyridine and 100 mM
imidazole at 25 C ........................................ 83

4-4 Dependence of the logarithm of kB(s"') on ApK, (the pK, of the zinc-bound
water subtracted from the pK. of the donor group) ...................... 93



















ix














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




PROTON TRANSFER IN CATALYSIS BY THE CARBONIC ANHYDRASES



By

J. Nicole Earnhardt

December 1998



Chairperson: Dr. David N. Silverman Major Department: Biochemistry and Molecular Biology

Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide to bicarbonate and a proton. This reaction requires proton transfer between the zinc-bound water at the active site and aqueous solution for each cycle of catalysis, and the most efficient isozymes facilitate this transfer by the proton shuttle residue, histidine 64. In this work, I have characterized the catalytic properties of a recombinant murine carbonic anhydrase VII (CA VII), using stopped-flow spectrophotometry and "S0 exchange measured by mass spectrometry. CA VII has steady-state constants similar to the most efficient isozymes of carbonic anhydrase, CA II and IV, and is strongly inhibited by the sulfonamides


x








ethoxzolamide and acetazolamide. The magnitude of k,, near 106 s', its pH profile, I0exchange data for both wild-type and a histidine 64 to alanine mutant, and inhibition by CuSO4 all suggest that His 64 is a proton transfer group in CA VII. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude compared with the full length counterpart. There is no change in k,/IKm which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers. This is the first evidence supporting a role of the amino-terminal end in enhancing proton transfer by any CA.

These studies of CA VII showed proton transfer capability even in the absence of His 64. The best example of proton transfer by a CA not having a histidine at position 64 is the mitochondrial isozyme, murine CA V (MCA V). MCA V has a sterically constrained tyrosine at position 64; it is not an effective proton shuttle, yet catalysis still achieves a maximal turnover in CO2 hydration of 3 x 105 s-1 at pH > 9. This study has identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contributes to proton transfer. Comparison of k, in catalysis upon replacement of Lys 91 and Tyr 131 with alanine yielded a reduction in catalytic activity of 50% from wild-type. The corresponding double mutant showed a strong antagonistic interaction between these sites suggesting a cooperative behavior in facilitating the proton transfer step of catalysis. These replacements caused relatively small changes in ku/K. indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.




xi








A wider range of pKa values for the proton donors was achieved by measuring enhancement of catalysis by imidazole, pyridine, and morpholine buffers in solution. These buffers enhance proton transfer steps in a mutant of MCA V, Y64A/F65A, to values similar to that of the intramolecular counterpart, histidine, in the mutant Y64H/F65A. The rate constants for proton transfer from these buffers to the zinc-bound hydroxide in catalysis of MCA V show a direct correlation to the difference in acid and base strength of the catalysts. Application of Marcus rate theory shows that this proton transfer has a small intrinsic energy barrier (near 0.8 kcal/mol), which is also characteristic of nonenzymic rapid proton transfer between nitrogen and oxygen acids and bases in solution. The Marcus parameters yield a large thermodynamic component (near 10 kcal/mol). This work is a counterpart to studies of proton transfer involving histidine 64 and identifies solvent and active-site reorganization as a dominant feature in proton transfer in catalysis by carbonic anhydrase.























xii













CHAPTER 1

INTRODUCTION


The Mammalian Carbonic Anhydrases


Carbonic anhydrases are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to form bicarbonate and a proton: CO2 + H204HCO3-+ H+

Carbonic anhydrase (CA) from animals, plants, archaebacteria and eubacteria have been classified into three gene families based on unrelated evolutionary histories. These are designated a, 3, and y (Hewett-Emmett and Tashian, 1996). The mammalian carbonic anhydrases are all included in the a-class and will be discussed in detail in the following pages. Physiological Function

There are at least seven functional mammalian isozymes of CA, referred to as CA I through CA VII. The different isozymes are found in various locations in the cell. Isozymes I, II, 11, and VII are all found in the cytosol. CA IV is a glycosylphosphatidylinositol (GPI)anchored membrane protein, CA V is found in the mitochondria and CA VI is a secretory protein (Table 1-1; reviewed in Dodgson, 1991 and Sly and Hu, 1995).

The isozymes of CA are distributed throughout the different body tissues and serve various physiological functions, all of which are associated with the reversible hydration of CO2 (reviewed in Dodgson, 1991 and Sly and Hu, 1995). For example, isozyme II,








2










Table 1-1: Cellular Location and Predominant Tissue Distribution of the Mammalian Carbonic Anhydrases.

Isozyme Cellular Location Main Tissue Distribution

CA I Cytosol Red cells

CA II Cytosol Ubiquitous, red cells and secretory tissues

CA III Cytosol Skeletal muscle and adipose tissue

CA IV Membrane-bound Ubiquitous, lung, and kidney

CA V Mitochondrial Liver and kidney

CA VI Secretory Salivary glands

CA VII Cytosol Salivary gland', brain, lung

Note: This information is reviewed in Dodgson, 1991 and Sly and Hu, 1995. Montgomery et al., 1991.
b Lakkis et al., 1997.
' Ling et al., 1994.








3

having the most diverse tissue distribution of all the carbonic anhydrases, provides such physiological functions as H' production for renal acidification of urine and gastric acid secretion, and HC03O for the production of pancreatic juice, saliva, ocular and cerebrospinal fluid. The involvement of isozyme II in the formation of aqueous humor has made this CA a target enzyme for the treatment of glaucoma (Maren, 1997). Sulfonamide inhibitors of CA are used to reduce the production of ocular fluid and thereby decrease intraocular pressure that is a condition of this disease. CA II is also involved in CO2 transport and/or exchange in the kidney, red cells and lung and CA IV provides this same function in lung, brain, skeletal and heart muscles. There is facilitated diffusion of CO2 in skeletal muscle by isozyme III, HCO3 reabsorption in the kidney by isozyme IV, and pH regulation by isozyme VI in the saliva. Isozyme V has a metabolic function of providing HC03- to carbamoyl phosphate synthetase I and pyruvate carboxylase for ureagenesis and gluconeogenesis, respectively. CA V has also been postulated to have a role in lipogenesis (Dodgson, 1991). Structure

Of the seven isozymes, crystal structures of human CA I (Kannan et al., 1975), II (Eriksson et al., 1988a; Hikansson et al., 1992), bovine III (Erickson and Liljas, 1993), human and murine IV (Stams et al., 1996; 1998) and murine V (Boriack-Sjodin et al., 1995) have been determined. All these isozymes have common features including nearly identical structures around the active site by comparison of their backbone atoms. A representative structure of human CA II is shown in Figure 1-1. Each isozyme is a spherical molecule with a 24 residue amino-terminal tail that is loosely fit in relation to the rest of the molecule as determined in crystal structures of isozymes I, II I and IV (the CA V crystal is of an amino-








4
















His 64


























Figure 1-1. A ribbon model of human carbonic anhydrase II determined from the crystal structure (Ikansson et al., 1992). The three histidine ligands of the zinc are 94, 96, and 119. The intramolecular proton shuttle residue, His 64, is indicated.








5

terminal truncated protein). These isozymes are structurally dominated by a central 10stranded twisting P-sheet with short helices located on the surface of the molecule.

The active site is located at the bottom of a central cavity approximately 15 A wide at the surface and 15 A deep. In the active site, a divalent zinc ion is coordinated to the nitrogen atoms of three histidine residues and a H20 molecule (Figure 1-1). The histidine ligands are held in a strict tetrahedral geometry through hydrogen bonds to a series of second-shell residues referred to as indirect ligands (Christianson and Fierke, 1996). The histidine and indirect ligands are conserved throughout the functional mammalian carbonic anhydrases with the exception of the indirect ligand, 244, which is hydrogen bonded to a histidine ligand by its main chain carbonyl group.

Another feature of the mammalian carbonic anhydrases that is strictly conserved and observed in each crystal structure is the hydrogen bonding system of two amino acid residues, Thr 199 and Glu 106, with the zinc-bound hydroxide (Figure 1-2). In this system the zincbound hydroxide is a hydrogen-bond donor to the side-chain hydroxyl of Thr 199, which in turn, is a hydrogen-bond donor to the carboxylate of Glu 106. The zinc-bond hydroxide also forms a hydrogen bond with another water molecule (referred to as the "deep water") located in a hydrophobic pocket in the active site and is hydrogen bonded to the amido group of Thr 199. This system apparently restricts the orientation of the zinc-bound hydroxide for efficient reaction with substrate CO2 (Merz, 1990; Krebs et al., 1993a; Liang et al., 1993a) and is important for binding of bicarbonate (Xue et al., 1993), sulfonamides and many anionic inhibitors (Erickson et al., 1988b; reviewed in Liljas et al., 1994).







6




Hydrophobic pocket

Thr 199
> N,1 C0- H Glu 106- "ELo .

Zinc
/-N
N

His96 N His l9


His 94













Figure 1-2. Residues near the active site of human carbonic anhydrase II. The three histidine ligands of the zinc are 94, 96, and 119. The two amino acids comprising the hydrogenbonded system are, Glu 106 and Thr 199. The line at the fight indicates the area of the hydrophobic pocket.








7

There is a hydrophobic and a hydrophilic area located towards the surface of the active site cavity and the difference in amino acids found in these regions result in the variations in the properties of the seven CA isozymes. Several studies suggest that CO2 is weakly bound in the active site and interacts with the hydrophobic area; yet, there is no crystal structure with detectable bound CO2 for verification nor has any one experiment specifically located the CO2 binding site (Figure 1-2; Krebs et al., 1993b). The residues that comprise this hydrophobic site are Val 121, Val 143, Leu 198 and Trp 209 in isozyme II (Alexander et al., 1991; Fierke et al., 1991). This hydrophobic cavity is also important for inhibitor binding as discussed below.

Last, in the active site of isozyme II there is an ordered array of hydrogen-bonded water molecules detectable through X-ray crystallography (Erickson et al., 1988a, Hikansson et al., 1992). This hydrogen-bonded water chain is necessary for proton transfer from the zinc-bound water to buffer in solution which can occur through a proton shuttle residue such as histidine 64 in isozyme II (Figure 1-1; Venkatasubban and Silverman, 1980; Tu et al., 1989a). This will be discussed in future sections. Inhibition

Inhibitors of CA include a variety of anions, neutral organic molecules, sulfonamides, and metal ions such as Cu(II) and Hg(II) (reviewed in Liljas et al., 1994). Competitive inhibitors with respect to CO2 in steady-state experiments include phenol in isozyme II and imidazole in isozyme I. Small anion inhibitors include, among many others, azide, thiocyanate, nitrate and formate. All inhibitors, with the exception of the dipositive metal ions mentioned above, are found to bind at or near the zinc ion within the hydrogen-bonded








8

system of the zinc-bound hydroxide, Glu 106 and Thr 199 (Figure 1-2). Almost all inhibitors appear to displace the deep water, most coordinate with the metal ion, and many contribute a hydrogen bond to the hydroxyl of Thr 199.

All aromatic and certain heterocyclic sulfonamides inhibit catalysis by binding to the zinc ion as anions with the nitrogen atom of the sulfonamide group, R-S02-NH- (reviewed in Liljas et al., 1994). More specifically, the NIT group replaces the zinc-bound water molecule and hydrogen bonds to the hydroxyl group of Thr 199 (Figure 1-2; Erickson et al., 1988b). One of the sulfonamide group oxygen atoms forms a hydrogen bond with the peptide NH of Thr 199 while displacing the deep water molecule and the second sulfonamide oxygen has no contact and is pointing away from the zinc. The sulfonamide is positioned in the hydrophobic pocket with van der Waals contacts to the hydrophobic residues and these interactions all depend on the substitutions in the aromatic ring of the sulfonamide. These hydrophobic residues lead to variations in the inhibitory properties of the various isozymes with respect to the sulfonamides.

Metal ions inhibit CA, especially the isozymes with histidine functioning as a proton shuttle. He' and Ct?' bind to the proton shuttle residue, His 64, in CA 11 and prevent proton transfer from this site (Tu et al., 198 1). Both nitrogens of the histidine are found to bind mercury at half occupancy in the crystal structure of isozyme 11 (Erickson et al., 1 988b).

Overview of the Catalysis


The enzymatic mechanism has been well studied in most of the mammalian carbonic anhydrases. The catalysis of the reversible hydration of carbon dioxide to form bicarbonate








9

and a proton occurs in two separate and distinct stages and is therefore referred to as a ping pong mechanism. The first stage of catalysis comprises the hydration of CO2 involving the direct nucleophilic attack of zinc-bound hydroxide on substrate CO2 to yield bicarbonate; the departure of bicarbonate leaves a water bound to the zinc (equation 1-1). The second stage requires a proton to be transferred from the zinc-bound water to buffer in solution (designated as B in equation 1-2) to regenerate the zinc-bound hydroxide (H*E indicates a protonated shuttle residue; Christianson and Fierke, 1996; Lindskog, 1997).


+ H20
CO2 + EZnOH" T EZnHCO3 ; HCO3 + EZnH2O (1-1)


EZnH20 + B ;=' H EZnOH. + B = EZnOH" + BH+ (1-2)


Interconversion of CO2 and HC03"

The steady state ratio kca,/K. contains rate constants for the steps from the binding of CO2 up to and including the first irreversible step in catalysis, which is the departure of HCO3 (Silverman and Lindskog, 1988). The carbonic anhydrases rapidly catalyze the conversion of CO2 to HCO3 as represented in the magnitude of the steady state rate constant kc,,,/K (Table 1-2). For isozyme II, the maximal value of k.fK. approaches that for the diffusion-controlled limit for the encounter rate of enzyme and substrate which is estimated at 109 to 1010 M suggesting that catalysis occurs as fast as CO2 can diffuse into the active site (Khalifah, 1973; Lindskog and Coleman, 1973).

For most of the mammalian carbonic anhydrases, the pH profile of ka$Km is described by a single ionization with a pKa near 7 with maximum activity at high pH. CA III is an








10







Table 1-2: Maximal Values of the Steady-State Constants with Values of Apparent pK for CO2 Hydration Catalyzed by the Mammalian Carbonic Anhydrases.

Isozyme kXl(. (x 10. M' s') pKakcav) kca, (x 10"5 s ) pK3kw)

Human CA I 5 7.0b 2 --a

Human CA 11a 15 7.1 14 7.1

Human CA 1Ic 0.03 <6.0 0.1 >8.5

Murine CA IVd 3.2 6.6 11 6.3 and 9.1

Murine CA V' 3 7.4 3 9.2

Rat CA VIf 1.6 -- 0.7 -Murine CA VII9 7.6 6.2 and 7.5 9.4 6.2 and 8.2

Note: Data obtained at 250C.
' Khalifah, 1971. pKx,.) is not available because k, is reported to increase with pH without reaching a plateau up to pH 8.7.
b Behravan et al., 1991. pKak.Wm) was determined from the pH dependence of the secondorder rate constant for the catalyzed hydrolysis of 4-nitrophenyl acetate. 'Jewell et al., 1991.
d Hurt et al., 1997.
Heck et al., 1994.
f Feldstein and Silverman, 1984. Data obtained at pH 7.5, and probably does not represent maximal values. Therefore, values of pKa are not yet determined. g Data obtained from this work (Chapter 2).








I1I

exception in that kA.K~ is described by a single ionization with a pI(3 near 5 (Table 1-2). This PKa found for ka/Km is associated with the ionization state of the zinc-bound water. Proton Transfer

The proton transfer step in the hydration direction of catalysis proceeds from the zincbound water to buffer in solution (equation 1-2). Both intramolecular proton transfer to a shuttle residue, such as His 64 in CA 11, and intermolecular proton transfer to small buffers that fit in the active site occur through a hydrogen-bonded water structure that surrounds the metal in the active site cavity. A network of hydrogen-bonded waters is observed in the crystal structure of CA II, and at least two intervening water molecules are found between the imidazole ring of the proton shuttle, His 64, and the zinc-bound water (Erickson et al., 1 988a). Consistent kinetic evidence for proton transfer along this network of hydrogenbonded waters has been obtained by studying the solvent hydrogen isotope effects on k, for the hydration of CO2 in CA 11. Here, two or more protons are determined to be in motion during intramolecular proton transfer (Venkatasubban and Silverman, 1980). Using sitedirected mutagenesis in isozyme 11 to place bulky residues in the active site at position 65 reduces proton transfer in catalysis and provides evidence for the disruption of this water structure (Jackman et al., 1996; Scolnick and Christianson, 1996). Proton transfer between water molecules is described by the "Grotthus chain mechanism" (Agmon, 1995). This mechanism refers to sequential proton transfer steps between water molecules which does not require the same proton to be transferred along the water chain. In addition, for the carbonic anhydrases, the first proton transfer from the zinc-bound water to the next adjacent water








12

molecule is postulated to form a hydronium-like ion and determines the rate of CA catalysis (Liang and Lipscomb, 1988).

Intramolecular proton transfer. The steady state constant k,, contains the rate constants from the enzyme-substrate complex to the end of catalysis; in CA this includes the proton transfer step to regenerate the zinc bound water (Silverman and Lindskog, 1988). Among the carbonic anhydrases, isozyme II has the greatest turnover number of 106 s' (Table 1-2; Khalifah, 1971). pH profiles of k, for this isozyme follow a titration curve with a pKa of 7, therefore, the catalytic rate, k.,, is dependent upon the ionization of a residue, or residues, with pK,'s near 7. The high rate of catalysis of isozyme 1I and the ionization observed in pH profiles of k,. is attributed to a histidine at position 64 that has been identified as the catalytic residue for intramolecular proton transfer (Tu et al., 1989a). Studies using isotope effects, pH dependencies, and chemical rescue have shown that these intramolecular proton transfer steps are rate-determining for maximal velocity (Silverman and Lindskog, 1988; Tu et al., 1989a). Therefore, the high rate of catalysis of the hydration of CO2 is determined almost entirely by the intramolecular proton transfer between His 64 and the zincbound water (Lindskog, 1984; Rowlett, 1984).

The role of position 64 in catalysis by carbonic anhydrase II has been well studied. The efficiency of His 64 as an acid-base catalyst in isozyme I is attributed to its pK, value of 7 which is similar to that of the zinc-bound water, and to optimal spatial location and environment in the active site. The location of the imidazole side chain of His 64 is 7 A from the zinc and it extends into the active-site cavity with no apparent interactions with other residues (Erickson et al., 1988a).








13

Among the other seven isozymes, CA IV and CA VII both have a histidine at position 64 and appropriately yield rate constants for proton transfer approaching that of the high efficiency isozyme II (Table 1-2; Hurt et al., 1997, Earnhardt et al., 1998a). However, it must be noted that His 64 is not the sole proton shuttle residue in these two isozymes as indicated by two ionizations in the pH profiles of k, (Table 1-2; discussed further in Chapter 2). In CA I a histidine at position 64 is present, however, it is not functioning as a proton shuttle (Behravan et al., 1991). Isozyme III has the slowest turnover number of 104 s- for the CA isozymes. The corresponding residue at position 64 in CA III, lysine, lacks significant intramolecular proton transfer capability and the proton shuttles residue(s) representing the ionization in k., remain unknown (Table 1-2; Jewell et al., 1991). However, in mutants of isozyme II where a histidine has been inserted at position 64 and 67, the maximal k,, values are restored to values closer to isozyme II (Jewel et al., 1991; Ren et al., 1995). Similar observations have been found for isozyme V with a tyrosine at position 64, and as a result turnover numbers do not approach those of isozyme II (discussed in Chapter 3, Heck et al., 1994; Earnhardt et al., 1998b). However, upon site-directed mutagenesis, isozyme II like properties are found in a Y64H/F65A mutant of isozyme V (Heck et al., 1996).

Intermolecular proton transfer. As described above, for the hydration of CO2 catalyzed by CA, buffer in solution is the final acceptor of protons that are transferred from the zinc-bound water. A buffer-dependent step in catalysis at low buffer concentrations is observed when the catalyzed initial velocity of CO2 hydration is determined at buffer concentration less than 10 mM for isozyme II (Silverman and Tu, 1975; Jonsson et al., 1976). By contrast, intramolecular proton transfer is found to be rate limiting at high buffer concentrations.








14

Buffer-mediated enhancements are observed in k,, for hydration of CO2 by CA II and are associated with the intermolecular proton transfer from the active site to solution. For the proton independent steps of equation 1- 1, no enhancements are observed in k.jKm upon addition of buffer. Under steady-state conditions in isozyme II, the rate constants for proton transfer from the enzyme to buffer in the catalyzed hydration of CO2 depend on the difference in pK. between the enzyme as proton donor and the buffer as acceptor, consistent with bimolecular proton transfer between nitrogen and oxygen acids and bases in solution (Rowlett and Silverman, 1982). In this work, the absence of a trend in the structure of buffers that transfer protons yields supporting evidence for proton acceptance from a shuttle residue on the enzyme, which is known to be His 64 in CA HI, instead of proton transfer directly with the zinc-bound water.

Studies of buffer enhancement under chemical equilibrium conditions in isozymes HI and III have also demonstrated that small buffers that fit in the active site can provide an intermolecular proton shuttle group from the zinc-bound water to solution by directly accepting protons through intervening water bridges (Silverman and Tu, 1975; Tu et al., 1990). Buffers that lead to enhancement include imidazole, pyridine, and morpholine buffers and their derivatives and the observed enhancements in catalysis depend on the concentration of buffer and the buffer's chemical properties. Bronsted Analysis

As discussed in the previous section, Rowlett and Silverman (1982) have correlated rate constants for intermolecular proton transfer to the difference in acid and base strength of the catalysts under steady state conditions, which is referred to as a Bronsted analysis.








15

Bronsted plots for intramolecular proton transfer in isozyme III have also been constructed. Using chemical equilibrium methods, the rate constants for proton transfer were determined from a series of mutants with a histidine or glutamates and aspartates as proton shuttles placed at position 64 (Silverman et al., 1993; Tu et al., 1998) and position 67 (Ren et al., 1995). Variations in the pKa of the acceptor group under these conditions, the zinc-bound water, were obtained by mutagenesis of an active site residue, Phe 198 to either Leu or Asp (LoGrasso et al., 1991, 1993). An increase upon introduction of these residues in pK. of the zinc-bound water is possibly due to a change in the interaction of the hydroxyl side-chain of Thr 199 with the zinc-bound water that is transmitted through these mutants (Chen et al., 1993).

The resulting Bronsted curves could be fit by the Marcus rate theory. This allows the energy required for proton transfer from either position 64 or 67 in these experiments to be determined in terms of the intrinsic kinetic barrier for proton transfer, which is found to be consistently low (1.3 to 2.2 kcallmol; Silverman et al., 1993; Ren et al., 1995; Tu et al., 1998). Solvent hydrogen isotope effects studied with small buffers in human CA II under steady-state conditions also exhibit a low intrinsic kinetic barrier to proton transfer (-1 kcallmol; Taoka et al., 1994). This low intrinsic kinetic barrier defines the energy required for proton transfer between nitrogen and oxygen acids and bases in CA and is similar in magnitude to the intrinsic kinetic barrier for bimolecular proton transfer between these two groups in solution (2 kcallmol; Silverman et al., 1993). However, the energy required to orient the protein and/or active site water for this efficient proton transfer, expressed as a work function in Marcus theory, is large, 10 kcallmol, and accounts for the slow rate of CA








16

catalysis (106 s-') when compared to that of proton transfer in excited states (102 s-) (Silverman et al., 1993; Ren et al., 1995). Proton transfer from the zinc-bound water to the proton shuttle group occurs through an active site water structure. Therefore, water is essential for proton transfer and may be involved in the efficiency of the carbonic anhydrases due to a required reorganization of the water lattice before proton transfer can occur. This type of analysis will be discussed in detail in work on isozyme V in Chapter 4.

In the coming chapters, a detailed description will be provided of the catalytic properties of a new isozyme of carbonic anhydrase, isozyme VII. This work will establish the intramolecular proton shuttle residue as histidine 64 in this isozyme and provide novel insight into the influence of the amino-terminus on proton transfer. Also, in future chapters the intramolecular proton shuttle residues will be identified that contribute to catalysis by the mitochondrial CA, isozyme V. These proton shuttles are located at more distant sites from the zinc than position 64. Last, chemical rescue experiments will be described that involve buffers in solution as intermolecular proton shuttles. Small imidazole, morpholine and pyridine type buffers enhance catalysis in isozyme V, an isozyme that lacks a single predominant proton shuttle such as histidine 64. Overall, this work will investigate intramolecular proton transfer from near and distant proton shuttles in isozymes V and VII and intermolecular proton transfer from small buffers that lacks any distance requirements in isozyme V. This discussion of carbonic anhydrase describes proton transfer in a very welldefined and accessible system. It is hoped that the results obtained in this study will be applicable to describe proton transfers in much more complex systems that almost surely involve proton transfer through intervening water molecules. These complex proton








17

translocation systems that are under intense current scrutiny include rhodopsin of visual pigment, cytochrome oxidase in cellular metabolism, and the photosynthetic reaction center of plants.













CHAPTER 2
THE CATALYTIC PROPERTIES OF MURINE CARBONIC ANHYDRASE VII


Introduction


Carbonic anhydrase VII has recently been discovered by gene isolation from a human genomic library using a mouse CA II cDNA clone as a probe (Montgomery et al., 1991). The gene structure of CA VII is found to be very similar to the other functional isozymes, and CA VII is postulated to be a cytosolic enzyme (Montgomery et al., 1991). Phylogenetic analysis based on the amino acid sequences of the carbonic anhydrase isozymes closely relates CA VII to the mitochondrial CA V (Hewett-Emmett and Tashian, 1996). Both CA V and CA VII genes map to human chromosome 16 (Montgomery et al., 1991; Nagao et al., 1993). CA VII mRNA has been detected in baboon salivary gland (Montgomery et al., 1991) and rat lung (Ling et al., 1994). A recent detailed in situ hybridization study of CA VII mRNA expression in adult mouse brain revealed a wide, nonspecific distribution in different regions of the cerebrum and cerebellum (Lakkis et al., 1997). CA VII has also been reported from a cDNA library prepared from multiple sclerosis lesions found in a human patient (GenBank Acc. No. N78377).

A nearly complete mouse CA VII cDNA obtained by RT-PCR using RNA isolated from adult mouse (C57/BL6) brain showed a protein sequence identity of about 95% with the human sequence; the nucleotide sequences are about 91% identical (Ling et al., 1995). It has 18








19

been suggested that this close conservation in two mammalian forms of CA VII is indicative of the functional importance of this isozyme (Lakkis et al., 1996). This evolutionary conservation, together with the fact that CA VII is seemingly expressed in a wide variety of tissues, albeit at low levels, suggests that it may have an important general function in most cells. For this reason, a study of its kinetic properties is well worth investigating.

Initial kinetic characterization of the expressed recombinant murine CA VII demonstrated an isozyme with rather low CO2 hydration activity between pH 6.5 and 8.2 when compared to bovine CA 11 (Lakkis et al., 1996). This chapter contains a more complete kinetic characterization of CA VII determined by stopped-flow spectrophotometry, "0 exchange using mass spectrometry at chemical equilibrium, hydrolysis of 4-nitrophenyl acetate, and inhibition by two sulfonamides and CuSO4. The results of these studies show a high activity enzyme with the maximal values of katKm and k for CO2 hydration approaching that of CA II, placing it in the subset of rapidly acting carbonic anhydrases that includes isozymes II and IV. The role of His 64 as a prominent proton shuttle in CA VII as in isozymes II and IV was verified upon analysis of the kinetic properties of a His 64 to Ala mutant of CA VII. Similar to murine CA IV (Hurt et al., 1997), evidence indicates that CA VII shows multiple intramolecular proton transfers involving the zinc-bound water and at least two residues that act as proton shuttles, one of which is His 64. A truncation mutant of CA VII lacking 23 residues at the amino-terminal end showed intramolecular proton transfer decreased by an order of magnitude while kKs, was unchanged, suggesting a role for the amino-terminal end in proton transfer to the active site. And finally, CA VII was found to have the strongest inhibition by the sulfonamides acetazolamide and ethoxzolamide for any mammalian carbonic anhydrase.








20

Materials and Methods


Expression and Purification of a Recombinant Murine CA VII cDNA

I received from the laboratory of Dr. Richard E. Tashian (University of Michigan) an almost entire recombinant murine CA VII cDNA (rMCA VII'). In Dr. Tashian's lab, Dr. Maha M. Lakkis obtained this cDNA by RT-PCR using RNA isolated from adult mouse (C57/BL6) brain (Lakkis et al., 1996). This PCR fragment was amplified using a human CA VII 5' primer and a mouse CA VII 3' primer (see Figure 2-1). It was then cloned into the glutathione S-transferase expression vector, pGEX.KG, a derivative of pGEX-2T (Guan and Dixon, 1991; Lakkis et al., 1996). Lakkis et al. (1996) determined from N-terminal sequencing that the expressed protein from the glutathione S-transferase expression system contains two extra amino acids at the amino terminal end from the thrombin cleavage site. The resulting plasmid, pGEXmCA7, was sent to us from Dr. Tashian's laboratory. pGEXmCA7 was transformed into Escherichia coli (DH5a) and rMCA VII protein expressed and purified as follows (Smith and Johnson, 1988; Guan and Dixon, 1991; Lakkis et al., 1996). IPTG was added to a final concentration of 0.4 mM to induce expression of rMCA VII in DH5a cells grown in 2 x YT medium (with ampicillin). Frozen DH5a cells containing the expressed rMCA VII were thawed in a solution of PBST containing 2 mM 1Abbreviations: rMCA VII, recombinant murine carbonic anhydrase VII; MCA VIIb, a truncated form of murine carbonic anhydrase VII lacking 23 residues from the amino-terminal end; Mes, 2-(N-morpholino) ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; Hepes, N-[2-hydroxyethyl]piperazine-N'-2-ethanesulfonic acid; Taps, 3[tris(hydroxymethyl)methyl] aminopropanesulfonic acid; Ches, 2-(cyclohexylamino) ethanesulfonic acid; IPTG, isopropyl-3-D-thiogalactoside; PBST is the solution containing 150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.3 and 1% Triton X-100; SHIE, solvent hydrogen isotope effect.














GGATCCATGACCGGCCACCACGGCTGGGGCTA C GC T
1-------------------------------- CGGCCAGGACGACGGCCCTTCAAATTGGCACAAGCTGTATCCCATTGCCCAGGGAGAC 90
----------------------------- GCCGGTCCTGCTGCCGGGAAGCGTAACCGTGTTCGACATAGGGTACGTCCCTCTG
-4 ? ? ? ? ? ? ?7? ? GQ 0D DG PS N WH K LY P I AQ G D 26
G S MTG H H G WG Y H H
rMCA VII 4 MCA VIIb4

A C T A G C
91 CGCCAGTCACCCATCAATATCATATCCAGCCAGGCTGTGTACTCGCCCAGCCTGCAGCCACTGGAACTTTTCTATGAGGCCTGATGTCC 180
GCGGTCAGTGGGTAGTTATAGTATAGGTCGGTCCGACACATGAGCGGGTCGGACGTCGGTGACCTTGAGATACTCCGACGTAAGG
27 R Q S P I N I I S S Q A V Y S P S L Q P L E L F Y E A C M4 S 56
S

A C C A
181 CTCAGCATCACCAACAATGGCCACTCTGTCCAGGTGGACTTCAATGACAGTGATGACAGAACCGTGGTGTCTGGGGGCCCCC 3GAGGG 270
GAGTCGTAGTGGTTGTTACCGGTGAGACAGGTCCACCTGAAGTTACTGTCACTACTGTCTTGGCACCACAGACCCCCGGGGGACCTTCCC
57 L S I T N N G H S V Q V D F N D S D D R T V V S G G P L E G 86
T

C T T A TG T T G
271 CCTTGCCACGTCCTCCGGCAAGGGAAGGTAACCCGGAGCATCTCCG 360
GGGATAGCGGAGTTCGTCGAGGTGAAGGTGACCCCGTTCTTCGCGCTGTACCCGAGTCTCGTGTGTCACCTCGTTCAGGGTCG
87 P Y R L K Q L H F H W G K K R D M G S E H T V D G K S F P S 116
F H V

G T CT A T
361 GACAACGTCCGACCAGATCGATTGGGGCGTCGCCGTGCGCGGTGTT 450
CTGTTGCAGGCTGGTCTAGCTAACCCCGCAGCGGCACGCGCCACAA
117 E L H L V H W N A K K Y S T F G E A A A A P D G L A V V G V 146
S

Figure 2- 1. The sequence of MCA VII cDNA and derived amino acids. Differences from the human sequence (Montgomery et al., 199 1) are indicated above and below the murine sequence. Numbering is based on the human CA I standard with the second N-terminal residue (Gly) as number 1. The four murine amino acids shown in bold italic were confirmed by direct sequencing of the expressed recombinant murine CA VUI protein LAkkis et al., 1996). Underlined nucleotides, identify the human and mouse primers used to amplify the recombinant MCA VII cDNA that expressed the full-length form of CA VII (rMCA VII -4). The P23M mutation beginning the truncated MCA VII sequence is indicated (MCA VI1b --#). His 64 is indicated in bold. N

















TT c c T T T G C G C G A
451 TTCCTGGAGACAGGAGATGAGCACCCAAGCATGAACCGCCTGACAGACGCCCTCTACATGGTTCGATTTAAGGACACCAAGGCCCAGTTC 540
AAGGACCTCTGTCCTCTACTCGTGGGTTCGTACTTGGCGGACTGTCTGCGGGAGATGTACCAAGCTAAATTCCTGTGGTTCCGGGTCAAG
147 F L E T G D E H P S M N R L T D A L Y M V R F K D T K A Q F 176
G

c TG C G T G T
541 AGCTGCTTCAACCCCAAGTGCCTGCTGCCCACCAGCCGGCACTACTGGACCTATCCTGGCTCCCTGACCACACCCCCACTCAGTGAGAGT 630
TCGACGAAGTTGGGGTTCACGGACGACGGGTGGTCGGCCGTGATGACCTGGATAGGACCGAGGGACTGGTGTGGGGGTGAGTCACTCTCA
177 S C F N P K C L L P T S R H Y W T Y P G S L T T P P L S E S 206
A

C C T C T A G G T G C
631 GTCACTTGGATTGTGCTTCGGGAGCCCATCAGGATCTCCGAGAGGCAGATGGAGAAATTCCGGAGCCTGCTTTTCACCTCAGAGGATGAT 720
CAGTGAACCTAACACGAAGCCCTCGGGTAGTCCTAGAGGCTCTCCGTCTACCTCTTTAAGGCCTCGGACGAAAAGTGGAGTCTCCTACTA
207 V T W I V L R E P I R I S E R Q M C K F R S L L F T S E D D 236
C G

C A A C A G C G
721 GAGAGGATCCATATGGTGGACAACTTCCGGCCACCACAGCCGCTGAAGGGCCGAGTGGTCAAAGCATCCTTCCAGGCCTGA 801
CTCTCCTAGGTATACCACCTGTTGAAGGCCGGTGGTGTCGGCGACTTCCCGGCTCACCAGTTTCGTAGGAAGGTCCGGACT
237 E R I H M V D N F R P P Q P L K G R V V R A S F Q A 262
N R







Figure 2-1 (continued)








23

EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 0.4 mM MgC2lz, 0.4 mM ZnSO4, 0.1% P-mercaptoethanol, 0.1 mg/mL deoxyribonuclease I and 0.5 mg/mL lysozyme with stirring for two hours at 4C. After cell lysis the cell debris was pelleted by centrifugation at 23,000 x g for 1 hour at 4C. The supernatant was subjected to two affinity chromatography steps. First, the supernatant containing the glutathione S-transferase/rMCA VII fusion protein was stirred with 10 mL swollen glutathione S-agarose beads (Sigma Chemical Company) and then equilibrated in PBST at 40C to allow binding of the fusion protein to the beads. The beads were washed with cold PBST and 20 mL of thrombin cleavage buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCI2, and 0.1% P3mercaptoethanol) was added to equilibrate the beads for thrombin cleavage. Thrombin cleavage of the fusion protein was achieved by the addition of 10 U of thrombin in thrombin cleavage buffer and incubation at room temperature for 30 min. rMCA VII was collected in the eluate and dialyzed against 1 mM Tris (pH 8.0). The second purification step involved affinity chromatography on a gel containing p-aminomethylbenzenesulfonamide coupled to agarose beads according to Khalifah et al. (1977) with minor modifications. The enzyme was stored at 4 C, -20 'C or -70 C for several months with 100% recovery of the original activity that was determined during its purification at 4 C. Subcloning and Site-Directed Mutagenesis of CA VII

P23M truncation and P23M/H64A truncation mutants of CA VII. A truncated CA VII and a truncated H64A CA VII mutant were prepared by Dr. Minzhang Qian in the laboratory of Dr. Philip J. Laipis (University of Florida) and their methods for producing these mutants are as follows. Oligonucleotides which introduced 1) an Nde I restriction site








24

containing a methionine codon at position 998 in pGEXmCA7, changing the Pro (CCC) codon at the cDNA amino acid position 23 to a Met codon (DS215: CACAAAGCTGCATA TGATTGCCCAGGG), and 2) a Bcl I restriction site (including the natural stop codon) at position 1728 in pGEXmCA7 (ATGGAGTCTTGATCAGGCCTGGAA) were synthesized and used in four separate PCR reactions to amplify the truncated form of murine CA VII (designated MCA VIIb throughout this chapter). This truncation removes the first 23 amino acids of the original rMCA VII cDNA. The products of the PCR reactions were cloned into pGEM-T (Promega) and four independent clones from the separate PCR reactions sequenced. Two clones (all four contained the correct sequence) were then mutated using single-stranded DNA template and a mutating oligonucleotide (AGAGGATTCACATGGTGGACA) to remove the naturally occurring Nde I site at position 724 in the rMCA VII cDNA insert (Kunkel et al., 1987). The mutated inserts were removed from pGEM-T by digestion with Nde I and Bcl I and inserted into a Nde I and BamH I cleaved pET3 I+ expression vector (Tanhauser et al., 1992) to allow efficient high-level expression and site-directed mutagenesis. This expression plasmid constructed for the truncation mutant is termed pET3 l+MCA7b. Construction of the His 64 to Ala mutant form of MCA VIIb, the truncated mutant, was as previously described using a mutating oligonucleotide and a uracil-containing single-strand template (Kunkel et al., 1987; Tanhauser et al., 1992). This expression plasmid constructed for the truncation mutant containing the His 64 to Ala mutation is termed pET3 I +MCA7b H64A.

H64A mutant of CA VII. A full-length wild-type CA VII that is lacking the two extra amino acids at the amino-terminal end of the protein that are normally produced by the








25

glutathione S-transferase expression system and the H64A mutant of full-length CA VII were prepared and placed in a pET3 I+ expression vector by Ms. Nina Wadhwa in the laboratory of Dr. Philip J. Laipis (University of Florida) and their methods for producing these mutants are as follows. An oligonucleotide (DS225: CGCGTGGACATATGACCGGCCATCAC) which introduced an Nde I restriction site containing a methionine codon at position 932 (the normal start position in the absence of the two thrombin cleavage residues) in pGEXMCA7 was synthesized and used with DS214 in separate PCR reactions to amplify the full-length form of rMCA VII. The products of the PCR reactions were cloned into pGEM-T (Promega) and individual clones from separate PCR reactions sequenced. Three individual clones with correct sequence were digested with Nde I and Pvu II, releasing a 471 bp fragment containing the first 157 amino acids of rMCA7. This fragment was cloned into a similarly digested pET3 I+MCA7b expression plasmid. This effectively replaced the truncated amino terminus of the MCA7b insert with the full length sequence while eliminating the necessity for both site-directed mutagenesis to remove the Nde I and Barn HI sites and sequencing of more than the first 471 nucleotides. This expression plasmid constructed for the full-length CA VII is termed pET3 1+MCA7. The His 64 to Ala mutant of the full-length rMCA VII (without the two thrombin cleavage residues) in the pET3 1+ system was derived from pET3 I+MCA7b H64A by removing a 274 bp Pst I (bp 638-912) fragment and inserting it into a dephosphorylated, Pst I digested pET3 1+MCA7 full-length backbone from which the corresponding fragment (704-978) had been removed.

The kinetic properties of rMCA VII expressed from the pET3 1+ expression vector, which lacks the two addition amino-terninal thrombin cleavage residues, was unchanged from








26

that of rMCA VII, which contains two extra amino acids at its amino-terminal end. Therefore, unless otherwise indicated rMCA VII protein expressed from the glutathione Stransferase expression system was used for the experiments in this chapter.

Expression and purification of CA VII proteins expressed from the pET3 I+ vector.

The various forms of rMCA VII and MCA VIIb were expressed in E. coli strain BL21(DE3)pLysS as described (Tanhauser et al., 1992). DNA from the expression plasmids used to produce each protein was sequenced to confirm the structure of each insert. Purification was performed through previously described procedures using affinity chromatography on a gel containing p-aminomethylbenzenesulfonamide coupled to agarose beads (Khalifah et al., 1977; Heck et al., 1994). The enzymes were then stored at 4C. Enzyme Purity

Electrophoresis on a 10/o polyacrylamide gel stained with Coomassie Blue was used to confirm the purity of all of the CA VII enzyme samples. All enzyme samples used in the kinetic experiments were greater than 95% pure. Active CA VII enzyme concentration was determined by inhibitor titration of the active site with ethoxzolamide (K, = 0.5 nM) by measuring "0 exchange between CO2 and water (see below). The molar absorptivity at 280 nm was determined to be 2.6 x 10' M cm-' for rMCA VII. Stopped-Flow Spectrophotometry

Initial velocities were determined by following the change in absorbance of a pH indicator (Khalifah, 1971) at 25 C using a stopped-flow spectrophotometer (Applied Photophysics Model SF. 17MV). CO2 solutions were made by bubbling carbon dioxide into water or D20 for the solvent hydrogen isotope effect studies. The maximum concentration








27

of CO2 in H20,O achieved by this method was 17 mM following dilution (Pocker and Bjorkquist, 1976). Dilutions were made through two syringes with a gas tight connection, the CO2 concentrations ranged from 1.7 to 17 mM in H20. For the dehydration direction, KHCO3 was dissolved in degassed water and the HCO," substrate concentrations ranged from 2.8 to 25 mM in H20. Final buffer concentrations were 25 mM and Na2SO,4 was used to achieve a final ionic strength of 0.2 M. The buffer-indicator pairs, pK, values and the wavelengths observed were as follows: Mes (pK, = 6.1) and chlorophenol red (pI. = 6.3) 574 nm; Mops (pI, = 7.2) andp-nitro phenol (pKa = 7.1) 401 nm; Hepes (pIK = 7.5) and phenol red (pK, = 7.5) 557 nm; Taps (pK, = 8.4) and m-cresol purple (pK., = 8.3) 578 nm; Ches (pIK = 9.3) and thymol blue (pK, = 8.9) 596 nm. For each substrate at each pH, the mean initial velocity was determined with at least 6 traces of the initial 5-10% of the reaction. The uncatalyzed rates were determined in a similar manner and subtracted from the total observed rates. The kinetic constants, kc., and k,/Kn, and the apparent ionization constants from their pH profiles were determined by a nonlinear least-squares methods using Enzfitter (ElsevierBiosoft). Standard errors in k,,and kl/Km were generally in the range of 5% to 20% and 1% to 10% respectively.

"SO Exchange

The rate of exchange of "O between species of CO2 and water (equations 2-1 and 22) is catalyzed by carbonic anhydrase (Silverman, 1982).

HCOO"O- + EZnH20 # EZnHCOO"O- + H20 # COO + EZn"OH" + H20 (2-1) kB + H20
EZn"OH + BH # H+EZn"OH'+ B EZnH2,"0 EZnH2O + H2,"O (2-2)
k.B








28

An Extrel EXM-200 mass spectrometer with a membrane permeable to gases was used to measure the exchanges of "0 shown in equations 2-1 and 2-2 at chemical equilibrium and 25 'C (Silverman, 1982). Solutions contained no added buffers and total ionic strength was maintained at 0.2 M with Na2SO4 unless otherwise indicated.

This method determines two rates in the catalytic pathway (Silverman, 1982). The first is R, the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Equation 2-3 shows the substrate dependence of R,.

R,/[E] = kCaZC[S]/(Keff S + [S]) (2-3)

Here [E] is the total enzyme concentration, k,,x is a rate constant for maximal HCOs" to CO2 interconversion, [S] is the substrate concentration of HCO;" and/or CO2, and Ksff is an apparent substrate binding constant (Simonsson et al., 1979). Equation 2-3 can be used to determine the values of k.xf/Ksffs when applied to the data for varying substrate concentration, or to determine kat/Ksffs directly from R,/[E] when [S] << Kffs. In the studies reported here, the values of R,/[E] as a function of total concentration of all species of CO2 was linear at ([CO2] + [HCO3-]) as large as 200 mM. This indicates that [S] << K~ffs and under this condition k, '/Kfrs can be obtained directly from 1/[E]. Under steady-state conditions, when [S] << K. all enzyme species are at their equilibrium concentrations. Hence in both theory and practice, k,Kf12 is equivalent to kc,/K, for CO2 hydration as measured by steady-state methods (Silverman, 1982; Simonsson et al., 1979). The kinetic constant kcjK. and the determination of apparent ionization constants from the pH profile were carried out by nonlinear least-squares methods using Enzfitter (Elsevier-Biosoft).

This method also determines a second rate which is the rate of release from the enzyme of water labeled with "0 designated RH0 (equation 2-2). A proton from a donor








29

group BH converts the zinc-bound 180-labeled hydroxide to zinc-bound 1120, which readily exchanges with unlabeled water and is greatly diluted into the solvent water H2160. The value of Rmo can be interpreted in terms of the rate constant from a predominant donor group to the zinc-bound hydroxide according to equation 2-4 (Silverman et al., 1993), in which kB is the rate constant for proton transfer to the zinc-bound hydroxide, KB is the ionization constant for the donor group and KE is the ionization constant of the zinc-bound water molecule. For our data RIo/[E] was determined by equation 2-4 using the program Enzfitter (ElsevierBiosoft).

RHo/[E] = kB/{(1 + KB/[H ])(1 + [H]/KE)} (2-4)

For solvent hydrogen isotope effects, RH2/[E] and kca/K were determined in 99.8% D20. All pH measurements are uncorrected pH meter readings. This is based on the assumption that the correction of a pH meter reading in 100% D20 to obtain pD (pD = pH + 0.4) is approximately canceled by the change of pKa in D20 for almost all acids with pKa between 3 and 10.

Inhibition

Inhibition by ethoxzolamide and acetazolamide was determined using "0 exchange at chemical equilibrium. The values of I. in Table 2-1 were obtained by least-squares fit of catalytic velocity to the expression for competitive inhibition as a function of inhibitor concentration under the conditions that the total substrate concentration ([CO2] + [HCO3] = 25 mM) was much less than the apparent binding constant for total substrate, I(.ffs. At the pH of these measurements, 7.3 to 7.5, KffS is greater than 100 mM for CA VII. The values of Y. determined from Rm0/[E] in the same manner agreed to within 25% with the values determined from R,/[E].








30












Table 2-1: Inhibition Constants IQ (Nanomolar) for Isozymes of Carbonic Anhydrase Determined by "O Exchange.

I, (nM)
Isozyme Acetazolamide Ethoxzolamide Reference

Human CA II 60 8 LoGrasso et al., (1991)

Human CA III 40,000 8,000 LoGrasso et al., (1991)

Murine CA IV ------ 16 Hurt et al., (1997)

Murine CA V 60 5 Heck et al., (1994)

Murine CA VII 16 0.5 This work

Note: All values of K were determined at pH 7.2-7.5 and 25 C.








31

Hydrolysis of 4-Nitrophenyl Acetate

Measurement of the esterase function of rMCA VII was performed by the method of Verpoorte et al. (1967) by following the absorbance change at 348 nm, the isosbestic point of 4-nitrophenol and its conjugate base, nitrophenolate ion. Concentrations of buffer and Na2SO4 were both 33 mM and initial velocities were determined at 25 *C. The uncatalyzed rates were subtracted from the observed rates, and the kinetic constant kj/Km and the determination of apparent ionization constants from the pH profile were carried out by nonlinear least-squares methods using Enzfitter (Elsevier-Biosoft).

Results


Recombinant Murine CA VII

The full length recombinant murine CA VII protein (rMCA VII) used in this study has a portion of its amino-terminal amino acid sequence derived from the human CA VII cDNA (Lakkis et al., 1996). More specifically, Ling et al. (1995) obtained a 91% complete mouse CA VII cDNA by RT-PCR using RNA isolated from adult mouse (C57/BL6) brain. This cDNA lacked sequence encoding the 28 amino-terminal residues. Lakkis et al. (1996) then constructed an almost complete murine CA VII cDNA by carrying out PCR under relatively stringent conditions with a 5' primer from the human CA VII sequence starting at the initial ATG and extending 26 nucleotides into the gene (Lakkis et al., 1996), and a 3' primer determined by the murine CA VII cDNA of Ling et al. (1995) (see Figure 2-1 for location of 5' and 3' primers). This construct allowed the determination of an additional 58 murine nucleotides after the 26 human 5' primer nucleotides at the 5' end of the murine CA VII cDNA (Figure 2-1). This extended murine CA VII cDNA sequence showed that 18 of the 19








32

newly-derived amino acids are identical between human and murine CA VII (Figure 2-1). However, the rMCA VII cDNA used in this study has 26 nucleotides (including the start ATG) that are of human origin (Figure 2-1) and thus encodes a chimeric CA VII protein with the 9 N-terminal amino acids of human origin and the remaining 253 from the mouse. The 9 amino-terminal residues of the murine CA VII protein remain unknown; however, in view of the highly conserved nature of the human and murine CA VII sequences, it is likely that they will be identical to the human sequence.

Catalytic Activity

The ratio k .JKm for rMCA VII determined from 0 exchange between CO2 and water yielded a pH profile that was best fit by the sum of two ionizations (Figure 2-2, Table 2-2; Tipton and Dixon, 1979). A very similar result to these was obtained by stopped-flow spectrophotometry in which the pH profile of ka/Km of rMCA VII for CO2 hydration over the pH range of 5.3 to 9.0 was also described by two ionizations with values of pKa of 6.2 0.9 and 7.6 0.5 and with a maximum of(3.3 1.0) x 107M-1 s"' at high pH (Data not shown). The maximal values of kca/K. for hydration measured by stopped-flow spectrophotometry were somewhat lower than those measured by "0 exchange. The results of the steady-state measurements of k,/Km for rMCA VII in the HC03" dehydration direction yielded a maximum at low pH and was dependent on a single ionizable group (Figure 2-2, Table 2-2). Because of the unfavorable equilibrium between CO2 and HCO3", the measurements in the dehydration direction were not extended to regions above pH 7.2. Hence, in the dehydration direction the second ionization at pH near 7.5 was not observed.

The pH dependence of k,/Km, determined from "0 exchange between CO2 and water, was also measured for the full-length rMCA VII H64A mutant. The pH profile of








33

108











II



5 7 80
!0
/













pH








Figure 2-2. The pH dependence of k,/K.1 for (0) hydration of CO2and (0) dehydration of HCO3- catalyzed by rMCA VUI at 25 *C. "~K~ for hydration was obtained by "0 exchange using solutions containing no buffers and in which the total ionic strength of solution was maintained at 0.2 M by addition of Na2SO4 and the total concentration of all species Of CO2 was 25 mMv. A nonlinear least-squares fit to the data points for CO2 hydration is represented by the solid line. The fit was to two ionizations with values of pI(, and maximal LJ/Km given in Table 2-2. The dashed line is a nonlinear least-squares fit to one ionization with a pI(, = 7.1 0. 1 and a maximal value of k.K at (7.2 0.3) x 10' MW s'. The ratio ku/Km for dehydration of HCO;- was obtained by stopped-flow spectrophotometry in the presence of 25 mM of one of the following buffers: pH 5.3-6.4, Mes; pH 6.6-7.2, Mops; pH 6.9-7.2, Hepes. Total ionic strength of solution was maintained at 0.2 M with Na2SO4. The solid line is a nonlinear least-squares fit of the data points to a single ionization with pI(8 and maximal k.X/K given in Table 2-2.








34








Table 2-2: Catalysis by Carbonic Anhydrase VII: Maximal Values of Steady-State Constants with Values of Apparent pKa.
k.X (MWs") pK4.r, ) km s pK4.m)


HydrationofCO2 (7.6 0.3) x 10' 6.2 0.5 (9.4 2.4) x 10 -8.2a
7.5 0.3 6.2 0.2

HydrationofCO2 (8.2 0.3) x 10 7.70.1 (4.5 0.4) x 105'
(MCA VIIb)b

Hydration ofCO2 (9.7 0.2) x 107 7.2 0.2 (1.6 0.2) x 105 8.9 0.2
(H64A)d
Hydrolysis of 71 8 5.3 0.3
4-Nitrophenyl Acetate 7.1 0.1

Dehydration of HCO3 (9.7 1.0)x 106 6.80.2 (1.90.1)x 105 6.70.1

Note: All data was obtained using rMCA VII protein except where indicated. Experimental conditions as given in the corresponding Figure legends. Because of the uncertainty in the maximal value of k, this pK, is poorly determined. b This truncated form of murine CA VII is lacking the amino-terminal 23 residues of rMCA VII shown in Figure 2-1.
Not measured. The maximal value of k, was determined from one measurement only, at pH
9.1. Therefore, a value of pK,(tlk,) could not be determined. d This is the H64A mutant of full-length rMCA VII.








35

kca/Km was described by one ionization (Table 2-2). A very similar result to these was obtained by stopped-flow spectrophotometry in which the pH profile of kc,/K. for CO2 hydration over the pH range of 5.9 to 9.5 was also described by one ionization with a value of pKa of 7.6 0.6 and with a maximum of(2.4 0.6) x 107M-1 s-' at high pH (Data not shown). The maximal value of kcjKm for hydration measured by stopped-flow spectrophotometry were somewhat lower than those measured by "0 exchange.

The pH dependence of ka,/Km for two truncated forms of murine CA VII were also measured from "0 exchange between CO2 and water. One truncated form had the aminoterminal 23 residues removed, with a new amino-terminus starting at the Pro 23 --, Met mutation; it is designated MCA VIIb (Figure 2-1). MCA VIIb was further mutated by replacing His 64 with Ala. MCA VIIb had values of k,.atKm for CO2 hydration adequately fit to a single pK with values given in Table 2-2. The pH profile of k,./Km for MCA VIIb H64A was identical.

Measurements of CO2 hydration by stopped-flow spectrophotometry gave a maximum value of k,, at pH > 9 for full-length rMCA VII and the H64A mutant (Figure 2-3, Table 22). The data for k, over the pH range of 5.3 to 9 was best fit to two ionizations for wildtype rMCA VII whereas the rMCA VII H64A mutant had a pH profile of k, described by one ionization (Figure 2-3, Table 2-2). Steady-state measurements for k, for dehydration of HC03 showed a maximum at low pH for wild-type rMCA VII (Figure 2-3, Table 2-2).

Values of the "O-exchange parameter RHo/[E] describe the proton-transfer dependent rate of exchange of H21O into solvent water (equation 2-2; Silverman, 1982). As was found for human CA II (Silverman et al., 1993), for rMCA VII the pH profile of Ro/[E]








36

107



106

105104



AA


103-A




102
5 6 7 8 9

pH







Figure 2-3. The turnover number k for (0) hydration of CO2 and (0) dehydration of HC03- catalyzed by rMCA VII and (A) hydration of CO2 catalyzed by rMCA VII H64A obtained by stopped-flow spectrophotometry at 25 TC in the presence of 25 mM of one of the following buffers: pH 5.3-6.4, Mes; pH 6.6-7.2, Mops; pH 6.9-7.5, Hepes; pH 7.7-8.3,
Taps; pH 8.6-9.1, Ches. Total ionic strength of solution was maintained at 0.2 M with Na2SO. The solid line is a nonlinear least-squares fit to two ionizations for the data points in CO2 hydration catalyzed by rMCA VII and for rMCA VII H64A the solid line is a nonlinear least-squares fit to one ionization with the value of pK. and maximal kJK. for both enzymes given in Table 2-2. The solid line through the data points for the dehydration of HC03- is a nonlinear least-squares fit one ionization with pK values and maximal k, also given in Table 2-2.








37

was bell-shaped with a maximum occurring near pH 7 (Figure 2-4). The H64A mutant of rMCA VII was found to have much reduced values of RH2o/[E] at pH < 8 as compared to rMCA VII (Figure 2-4). The inset of Figure 2-4 shows the pH profile for the differences in RuJ[E] between full-length rMCA VII and rMCA VII H64A. The shape and magnitude of this difference plot reflects the loss of a proton donor or donors of pK, near 7.1.

R1m/[E] for rMCA VII was inhibited by CuSO4 with an inhibition constant of 0.33 iiM at pH 7.5 and 25 C; the addition of CuSO4, up to a final concentration of 40 PiM, to rMCA VII had no effect on R,/[E] (Data not shown).

The truncation mutants of CA VII had much reduced values of RHo/[E] at pH < 8 (Figure 2-5). The values of Rm[E] catalyzed by truncated MCA VIIb H64A were reduced even further in this region of pH (Figure 2-5). It must be noted that in Figure 2-5 the data of Ram[E] for MCA VIb H64A was not adequately fit to a single ionization at pH less than 7. This deviation of the data from a single ionization behavior may reflect enzyme denaturation at low pH or perhaps the removal of the amino terminus and histidine 64 has generated a mutant with a more complex pH profile of RI /[EI at low pH. If this is the case, then this poor fit is a result of the lack of consideration of other influences or ionizations that may contribute to the pH dependence of RH2o/[E] for this mutant. In either case, the inset of Figure 2-5 shows the pH profile for the differences in RH2/[E] between the full-length rMCA VII and the truncated MCA VIlb, and between MCA VIIb and MCA VIlb H64A. The shape of these two difference plots is very similar reflecting in each case the loss of a proton donor or donors of pK near 6.9 to 7.5.








38
10s 3
106 Difference RH20/[EJ

(x 105 S) 2





00
05105 6 7 8 9

MOM"


e 4
104





1031,,
5 6 7 8 9
pH


Figure 2-4. Variation with pH of Rmo/[E], the proton-transfer dependent rate constant for the release from the enzyme of "O-labeled water, catalyzed by (@) rMCA VII and (A) the H64A mutant of rMCA VII. Solutions contained no buffers and the total ionic strength of solution was maintained at 0.2 M by addition of Na2SO4; the total concentration of all species of CO2 was 25 mM. The solid line is a fit of equation 2-4 to the data for rMCA VII with values of pK. for the proton donor of 7.4 0.1 and acceptor groups of 5.8 0.1 and i4, the rate constant for proton transfer to the zinc-bound hydroxide in the dehydration direction of catalysis, of(3.2 0.4) x 10 sf'. The solid line representing the fit of equation 2-4 to the data for rMCA VII H64A yielded pK, values for the proton donor of> 9 and acceptor groups of 6.5 0.1 and 14 = (2.6 0.1) x I0 s'. Inset: The difference in Rmo/[E] between rMCA VII and rMCA VII H64A. The solid line is a nonlinear least-squares fit describing proton transfer (equation 2-4) from a donor group of pK, 7.1 0.1 and zinc-bound hydroxide the conjugate acid of which has pK. 5.9 0.1 with a rate constant for proton transfer k4 = (3.3 0.3) x 10' s-'. Note, at pH > 8.5 the fit of the data for wild-type and H64A cross. This is a result of the fit of rMCA VII to only two ionizations and our lack of consideration of the addition contribution of a proton donor group at pH > 8.5, that most certainly exists in the wild-type.







39

107 2
Difference RH2o/[E]

(x 10" -1) 1
106


00
1 6 7 8





105
103





102,
5 6 7 8 9

pH







Figure 2-5. The variation with pH of Rmo/[E] catalyzed by (0) full-length rMCA VII, (A) the H64A mutant of full-length rMCA VII, (0) the truncated MCA VIlb (see Figure 2-1), and (U) a H64A mutant of the truncated MCA VLIb. Experimental conditions are as described in Figure 2-4. Inset: (0) The difference in R,/d[E] between rMCA VII and the truncated form MCA VIIb. The solid line is a nonlinear least-squares fit describing proton transfer (equation 2-4) from a donor group of pK 6.9 0.2 and zinc-bound hydroxide the conjugate acid of which has pK, 5.9 0.2 with a rate constant for proton transfer of k. = (3.4 0.8) x 10' s. (N) The difference in Rmo/[E] between MCA VIlb and the H64A mutant of MCA Vfb. The solid line is a nonlinear least-squares fit describing proton transfer (equation 2-4) from a donor group of pK. 7.5 0.2 and zinc-bound hydroxide the conjugate acid of which has pK(, 6.2 0.2 with a rate constant for proton transfer of kB = (5.5 1.1) x I04 s








40

rMCA VII is able to catalyze the hydrolysis of 4-nitrophenyl acetate. The pH profile of kUK0 has a maximum at pH > 9 and is best fit to two ionizations (Figure 2-6, Table 2-2) similar to k/K0, in the CO2 hydration direction. In some isozymes of CA there is a nonspecific esterase activity not associated with the zinc, as has been found in CA I (Wells et al., 1975) and CA III (Tu et al., 1986). However, that is not the case for rMCA VII; at a variety of different pH values the esterase activity was found to be greater than 98% inhibited in the presence of equimolar concentrations of the active-site inhibitor ethoxzolamide (., =

0.5 nm) and enzyme (present at 10-' M).

Inhibition of "O exchange between CO2 and water catalyzed by rMCA VII with two classic sulfonamides acetazolamide and ethoxzolamide was tested. The resulting values of the inhibition constant Y, are compared to the inhibition values of other isozymes of CA in Table 2-1.

The solvent hydrogen isotope effects (SHIE) observed for catalysis by rMCA VII were 1.0 0.1 for k,/K. for CO2 hydration at pH 6.8. On k, the SHIE was 3.0 0.1 at pH 6.8 in solutions containing 25 mM Hepes consistent with rate-determining proton transfer involving the aqueous ligand of the zinc (equation 1-2, Chapter 1, page 9). The SHIE at pH

6.8 on RHo/[E] was 3.3 + 0.4.

Discussion


Comparison of Isozmes of Carbonic Anhydrase

I have compared the steady-state catalytic constants of CA VII with CA II, the most efficient of the carbonic anhydrase isozymes, and with five other isozymes in the a-class of








41


102











P2




101



5 6 7 8 9

pH













Figure 2-6. The pH dependence of k.IXm for the hydrolysis of 4-nitrophenyl acetate catalyzed by rMCA VII. Data were obtained at 25 C in 33 mM of one of the following buffers: pH 5.3-6.5, Mes; pH 6.9-7.2, Mops; pH 7.3-7.7, Hepes; pH 8.1-8.9, Taps; pH 9.19.4, Ches. The solid line is a nonlinear least-squares fit of two ionizations with pK. values and maximal k.XK given in Table 2-2.








42

the carbonic anhydrases. The steady-state constant k,,/Km contains the rate constants up to and including the first irreversible step, which is the departure of HC03-; these are the steps of equation 1-1 (Chapter 1, page 9). The pH dependence of k.K describes the ionization state of the zinc-bound water (Christianson and Fierke, 1996; Lindskog, 1997). In the CO2 hydration direction, the maximal value of lJkm of 7.6 x 107 M" s' for rMCA VII is half that of CA II but somewhat greater than those for CA I, CA IV, and CA V (Table 1-2, Chapter 1, page 10). The observed solvent hydrogen isotope effect of 1.0 0.1 on kjKm indicates no rate-contributing proton transfer in the steps of equation 1-1 (Chapter 1, page 9) and is consistent with a direct nucleophilic attack of the zinc-bound hydroxide on CO2 (Lindskog, 1997); in this respect rMCA VII is similar to CA II and the other isozymes in the a-class.

The maximal value of the turnover number k., for hydration of CO2 catalyzed by rMCA VII approaches that of CA II (Table 1-2, Chapter 1, page 10). The value of k., contains rate constants for the steps from the enzyme-substrate complex through the proton transfers of equation 1-2 (Chapter 1, page 9). The kinetic constants for rMCA VII place it among the most efficient of the carbonic anhydrases with 67% of the activity of CA II. Considering the similarity in steady-state constants for CO2 hydration catalyzed by rMCA VII and CA II, it is interesting that the capacity of rMCA VII to catalyze the hydrolysis of 4nitrophenyl acetate (maximal k./K. = 71 Ms', Figure 2-6, Table 2-2) is much less than that of human CA II (maximal k/Km= 3 x 103 M's'; (Steiner et al., 1975)). Proton Transfer in CA VII

Histidine 64. Several results suggest that His 64 is the predominant proton shuttle in CA VII, as it is in CA II. First, there is no other residue in the active-site cavity which is a








43

likely shuttle of pK, near 7. Second, inhibition by CuSO4 of "0 exchange catalyzed by rMCA VII shows properties very similar to those observed for inhibition by CuSO4 of HCA II in which His 64 is a proton shuttle (Tu et al., 1981). In HCA II cupric ion coordinates to the imidazole side chain of His 64 and blocks the proton transfer role of this residue (Eriksson et al., 1986). This results in inhibition of RH2/[E] which is dependent on proton transfer, but has no effect on Rj/[E] which measures interconversion of CO2 and HCO3- in the first stage of catalysis (equation 2-1). The same pattern is seen for rMCA VII. Third, the pH profile for rMCA VII in Figure 2-4 is nearly identical with that of human CA II. And finally, as in the case of CA II (Tu et al., 1989a), replacement of His 64 by Ala has removed a predominant proton shuttle in CA VII, this result is described below.

The magnitude of k,,K. for hydration was found to be unchanged between rMCA VII and the H64A mutant of rMCA VII (Figure 2-2; Table 2-2). Thus, the rMCA VII H64A mutant is not affecting the catalysis of the interconversion of CO2 and bicarbonate at the zinc (equation 1-1, Chapter 1, page 9). Yet, there is an absence of one of the two ionizations in kca/K near 6 that was observed in wild-type rMCA VII. This maybe interpreted as the removal of an influence on the ionization state of the zinc-bound water by His 64 upon mutation of the histidine at position 64 to alanine (see discussion beginning on page 46). In contrast, both k, and Rmo/[E] are significantly reduced for H64A compared with wild-type (Figure 2-3 and 2-4; Table 2-2). Also, there is a loss of one of the two ionizations in k, and the difference between the pH profile for RH0/[E] for rMCA VII and for rMCA VII H64A (inset in Figure 2-4) shows the bell-shaped pH dependence consistent with the loss of a single proton shuttle of pKa 7.1 0.1, and corresponding to a loss in proton transfer capacity








44

(RH~[E]) of (3.3 0.3) x 105 s-1 upon mutation of His 64 to Ala. By this argument, the maximum near pH 6 in the pH profile for the rMCA VII is due to the function of His 64 as a proton shuttle (Figure 2-4). These results suggest that the H64A mutant is affecting proton transfer and that histidine is the predominant proton shuttle at position 64 in CA VII.

Effect of the am-ino-terminal residues. The maximal values of k,/Km and apparent pK.'s are the same for rMCA VII and for the truncation mutant MCA VIlb (Table 2-2). Thus, the amino-terminus has no role in and is not affecting the steps of equation 1-1 (Chapter 1, page 9), the catalysis by CA VII of the conversion Of C02 into bicarbonate at the zinc. However, two results suggest that the truncation is affecting proton transfer: the twofold decrease observed at pH 9.1 in k., for hydration compared with full length (Table 2-2), and the decrease by an order of magnitude in RH20I[E] near pH 6 (Figure 2-5). Aronsson et al. (1995) observed catalysis by a truncated variant of human CA II in which 20 residues at the amino terminus were removed; it had an overall CO2 hydration activity 15% of wild-type human CA IL.

Thus, it is reasonable to ask whether the truncation of the amino-terminal 23 residues has decreased the ability of His 64 to function as a proton shuttle. This suggestion is supported by the difference between the pH profile for RH2J1[E] for rMCA VII and for the truncated form MCA VIIb shown in the inset in Figure 2-5. This difference plot shows the bell-shaped pH dependence consistent with the loss of a single proton shuttle of pK, 6.9 0.2, and corresponding to a loss in proton transfer capacity (RH2C[E]) of 3.4 x 105 s-' upon truncation, similar to the removal of H64A from wild-type rMCA VII (see the difference plot in Figure 2-4). By this argument, the small maximum near pH 6 in the pH profile for the








45

truncated mutant MCA VIlb is due to the reduced capacity of His 64 to act as a proton shuttle (Figure 2-5).

The full removal of His 64 in the truncation mutant (H64A MCA VIIb) results in a change in the pH dependence when compared to the truncation mutant alone (Figure 2-5). The difference in values of RdJ[E] for MCA VIIb and H64A MCA VIIb is also given in the inset in Figure 2-5. Here again, the data are consistent with the loss of a single proton donor of pI(2 7.5 0.2 corresponding now to a smaller loss in proton transfer capacity of 5 x 104

g-1.

By this explanation the proton shuttle capacity of His 64 is lessened by removal of the amino-termninal 23 residues of rMCA VII. There are several possibilities for this loss. The first is a conformational change of the truncated form MCA VIIb in which His 64 is at a distance less effective for proton transfer or in which His 64 has a broader range of side-chain conformations than in the full-length enzyme and hence spends less time in the conformations appropriate for proton transfer. It is also possible that the three histidines, of the aminoterminal 23 residues (Figure 2-1) are the significant proton donors in rMCA VII. This is unlikely because based on the structure of CA 1I (Eriksson et al., 1988b) the distance of these residues from the zinc (> 18 A) is much greater than for His 64 (-7 A) and therefore this distance would not be considered optimal for significant proton transfer; moreover, it is His 64 that has been shown to play the predominant role as proton shuttle in rMCA VII as discussed above and in isozyme IL. It is possible however, that these three histidines could play a role as secondary proton shuttles in a proton relay mechanism with His 64 since there is an obvious difference upon truncation of the H64A mutant (MCA VIIb H64A) when








46

compared to the full-length H64A (rMCA VII H64A) in the pH profiles of RH2IJJ[E] at pH near 6 (Figure 2-5). Interestingly, it is found that deletions of amino-terminal residues of carbonic anhydrases that lack an effective proton shuttle at position 64, namely CA 1112 and CA V (Heck et al., 1994), show catalytic properties nearly identical with their full-length counterparts. This finding may support the suggestion that the amino-terminal end of rMCA VII influences the function of His 64 as proton shuttle. This topic is under further study.

Basic residues. Several results suggest there is another residue(s) in the active site of CA VII, in addition to His 64, that is participating as a proton shuttle in catalysis. In pH profiles of k,. for both wild-type and the H64A mutant there is an ionization at high pH (Table 2-2, Figure 2-2). Also, in pH profiles of RH0J[E] for full-length H64A and the truncated H64A mutant, where the capacity of His 64 to function as a proton shuttle group is removed, values of R,,J[E] plateau near 3-6 x I O's' at pH > 8. This plateau at high pH represents proton transfer from a donor group of pK. > 9 to the zinc-bound hydroxide in the dehydration direction of catalysis (Figure 2-4 and 2-5). The identity of the proton shuttle residue(s) that is contributing to catalysis at high pH is one focus of my work in isozyme V and the results of that work will be discussed in Chapter 3. Assignment of pK, Values

The pH dependence of k.JK., in the CO2 hydration direction can be described by two ionizations for rMCA VII, near PKa 6.2 and 7.5 (Figure 2-2, Table 2-2). The pH rate profile for the esterase activity of rMCA VII also appears dependent on two ionizations with similar values of pK. (Figure 2-6, Table 2-2). One of these ionizations is clearly that of the zinc2Hevia, A., Tu. C. K., Silverman, D. N., and Laipis, P. J., unpublished observations








47

bound water; the other ionization most likely results from a perturbation of the PKa of the zinc-bound water caused by the electrostatic interaction of a nearby group. Perturbations on the pK, of the zinc-bound water have been described for mutants of CA. For example, in human CAlR the introduction of a glutamate (Forsman et al., 1988) or a histidine (Behravan et al., 1991) in the active site changes the pH dependence of k./K. for ester hydrolysis from a single ionization to one described by two ionizations.

By comparing data from steady-state measurements for wild-type and mutants in CA VII an assignment of these two ionizations in pH profiles of k.jK. for rMCA VII has been achieved. Upon mutation of His 64 to Ala in rMCA VII the ionization near 6 disappears in pH profiles of k.fKm., leaving the second ionization near 7.5 (Figure 2-2; Table 2-2). This is also the case for the two truncation mutants of CA VII, MCA VIII, and MCA VIIb H64A, where only an ionization near 7.5 persisted in pH profiles of kJ/K. (Table 2-2). These results suggest His 64 perturbs the pKa of the zinc to yield the second ionization near pH 6.

The pH profile for k,. for hydration of CO2 catalyzed by rMCA VII also contains two ionizations, one with a pKa of 6.2 0.2 and one with a poorly determined pK, near 8.2 (Figure 2-3; Table 2-2). One of these ionizations is His 64 and the other ionization should represent some other proton shuttle(s) in the active site. The pH profile for k,,i for the H64A mutant is lacking an ionization at pH 6, and the ionization at high pH remains, which is in agreement with our assignment of the pKa value for His 64 near 6 in pH profiles of k,/K. as described above. The observations in rMCA VII of two ionizations influencing k., for hydration are very similar to the observations of Hurt et al. (1997) in which k, for murine CA IV was found to depend on two ionizations. One of these had an apparent pK. of 6.3 and was








48

assigned to His 64 upon analysis of the H64A mutant, and a second with pK, of 9.1 may represent proton transfer from basic residues more distant from the zinc than His 64; this result is observed for other isozymes of CA and is extensively discussed in Chapter 3 (Earnhardt et al., 1998b; Silverman et al., 1998).

The bell-shaped pH profiles typically found for RHo/[E] can be described by equation 2-4 which expresses RH2/[E] as the product of the protonated form of the donor group and the unprotonated form of the aqueous ligand of the zinc (Silverman et al., 1993). By this analysis, the data of Figure 2-4 for unmodified rMCA VII demonstrate two values of pK,, near 5.8 and 7.4, which again suggest the same ionizations as observed in k.K,, for CO2 hydration and hydrolysis of 4-nitrophenyl acetate (Table 2-2). However, the "0-exchange data of Figure 2-4 taken alone are equally consistent with the following two assignments: 1) The pKa of 7.4 is the zinc-bound water and the pK, of 5.8 is His 64 with kB = (1.3 0.4) x 10' s' for the rate constant for intramolecular proton transfer in the dehydration direction (as shown for "0 exchange in equation 2-2); 2) The pK of 7.4 is for His 64 and pK, of 5.8 is the zinc-bound water in which case kB = (3.2 0.4) x 10' s"-. Assigning the lower ionization near 6 to His 64 in the pH profile of RHo/[E] for rMCA VII yields a rate constant for proton transfer an order of magnitude higher than the two other fast isozymes, CA II and IV (Hurt et al., 1997; Tu et al., 1989a). Therefore, it is helpful to compare the "0-exchange data with the steady-state turnover for dehydration. Like kB, the turnover number for dehydration, k., is dependent on the proton transfer to the zinc-bound hydroxide. The maximal value of k, for dehydration catalyzed by rMCA VII was 2 x 10' s-' (Table 2-2), a value consistent with assigning the pK, near 7.4 to His 64 and the pK, near 5.8 to the zinc-bound water. This








49

assignment of His 64 in pH profiles of RH2o/[E] to 7.4 is supported upon comparison to pH profiles of rMCA VII H64A and the truncation mutant MCA VIIb, both mutants are lacking a predominant proton shuttle and are missing the pK of 7.4 and yield a pKa for the zincbound water nearer to 6. However, there is a discrepancy in assigning the pKa values of His 64 and the zinc-bound water in R,2[E], in that for steady state conditions it is the lower pKI near 6.2 that represents His 64 as described in the previous paragraphs. Further experiments are needed to understand this difference and provide a better interpretation. Inhibition

The inhibition of rMCA VII by the sulfonamides acetazolamide and ethoxzolamide measured by "S0 exchange is greater than for the other isozymes (Table 2-1). These sulfonamide inhibitors are expected to bind directly to the zinc and adhere to the hydrophobic side of the active-site cavity as demonstrated in human CA II (Eriksson et al., 1988b). Many of the residues implicated in sulfonamide binding with CA II are conserved in rMCA VII. Two exceptions are L204 and C206 in CA II which in murine CA VII are serines. Conclusions

Murine CA VII is a highly conserved isozyme of carbonic anhydrase and this conservation may be indicative of its functional importance. It is a very efficient carbonic anhydrase with catalytic activity 100-fold greater in kct over the slowest CA, isozyme III. It is therefore similar to the most active of the mammalian carbonic anhydrases, isozymes II and IV. Moreover, among these isozymes it is the most inhibited by two widely-used sulfonamides when measured by gO exchange. This indicates a highly specific interaction of inhibitors with CA VII that should be pursued by X-ray crystallography of the E.I complex.








50

Unique upon comparison to the other wild-type mammalian isozymes, CA VII demonstrates the effect of two ionizations in the pH profile of k.,fKm. This suggests a close interaction between the zinc and His 64 that is missing in other carbonic anhydrases and may contribute to rapid proton transfer. One of these is the ionization of the zinc-bound water (pK. 7.5) and the second is suggested to be His 64 (pK. 6.2). Moreover, for the first time a role for the amino-terminal end in enhancing proton transfer has been determined in catalysis for a carbonic anhydrase. More specifically, the amino terminus may be restricting His 64 to useful conformations for proton transfer.














CHAPTER 3
INTRAMOLECULAR PROTON TRANSFER FROM MULTIPLE SITES IN
CATALYSIS BY MURINE CARBONIC ANHYDRASE V


Introduction


Carbonic anhydrase V (CA V) is a mitochondrial enzyme found predominantly in liver (Table 1-1, Chapter 1, page 2); it is a member of the a class of carbonic anhydrases which includes the mammalian isozymes (reviewed in Dodgson, 1991). The catalytic properties of murine carbonic anhydrase V (MCA V)3 have been characterized (Heck et al., 1994) and its crystal structure is determined to 2.45-A resolution (Boriack-Sjodin et al., 1995). Similar to the other carbonic anhydrases of the a class, MCA V is a monomeric zinc metalloenzyme of molecular mass near 30 kDa that catalyzes the hydration of carbon dioxide to form bicarbonate and a proton. The catalytic pathway of MCA V is similar to that of the wellstudied CA II in many respects. The two stage catalysis of these isozymes is described in detail in Chapter 1 (page 9).

For CA II, proton transfer proceeds through an intramolecular proton shuttle (designated in equation 1-2 as H' to the left of E, Chapter 1, page 9) which subsequently releases the proton to solution. In CA II this intramolecular proton shuttle has been identified



3Abbreviations: MCA V, murine CA V; HCA II, human carbonic anhydrase II; Y64A, the mutant with Tyr 64 replaced by Ala; Tris, tris(hydroxymethyl) amino methane; Ted, 1,4 diazabicyclo [2.2.2] octane or triethylenediamine.

51









52

as His 64 (Steiner et al., 1975; Tu et al., 1989a) which extends into the active site cavity with the N8 of its imnidazole ring 8.2 A from the zinc and with no apparent interactions with other residues (Eriksson et al., 1988b). MCA V has a tyrosine residue at position 64 which is not an efficient proton shuttle (Heck et al., 1994). However, it is possible to activate MCA V by placing a histidine residue at position 64 along with other changes in the active site (Heck et al., 1996). Studies using isotope effects, pH dependencies, and chemical rescue have shown that these intramolecular proton transfer steps are rate-determining for maximal velocity (Silverman and Lindskog, 1988; Tu et al., 1989a).

Position 64 in carbonic anhydrase is not the only site from which proton transfer can occur. Liang et al. (1993b) placed a histidine residue at four other positions within the activesite cavity of isozyme II and found that a His at 67 was capable of enhancing catalytic activity but at a level no greater than 20%/ of the wild-type enzyme. Ren et al. (1995) showed that His 67 in human CA III is capable of proton transfer but again not as efficiently as His 64 in CA Ill. It is significant that the mutants of carbonic anhydrase lacking a histidine proton shuttle in the active-site cavity can still sustain a catalytic turnover k,, for hydration at pH near 9 of 10' s' as for human CA [II (Jewell et al., 199 1) and as great as 3 x I0 s !' for MCA V (Heck et al., 1994). This suggests the presence of one or more basic residues that act as proton shuttles.

Since MCA V supports catalysis at a rapid rate at high pH, but is lacking His 64 as a prominent proton shuttle, it is pertinent to ask what residues in MCA V support this significant activity. There are a number of lysine and tyrosine residues in MCA V located in the active-site cavity or around its rim. In this study these residues have been replaced with








53

alanine and the initial velocities in catalysis by the resulting mutants were measured using stopped-flow spectrophotometry; catalysis of "0 exchange between CO2 and water was also measured using mass spectrometry. The results show that the catalytic activity in MCA V is supported by multiple proton transfers involving a number of ionizable groups of basic pK, some more distant from the zinc than residue 64. Although there is no single prominent proton shuttle, Lys 91 and Tyr 131 with their amino and phenolic hydroxyl groups 14.4 A and 9.1 A from the zinc, as shown in Figure 3-1 (Boriack-Sjodin et al., 1995), account for about half of the catalytic turnover. Moreover, the interaction between these proton shuttles in catalysis is not simply additive, but antagonistic reflecting their adjacent location and suggesting a cooperative behavior in facilitating the proton transfer step of catalysis. Replacing four of these possible proton shuttle residues produced a multiple mutant that has 10% of the catalytic turnover k,, of the wild-type, suggesting that the main proton shuttles have been accounted for in MCA V. As a control, the replacements were determined to cause relatively small changes in k./K. for hydration which measures the interconversion of CO2 and HC03- in a stage of catalysis that is separate and distinct from the proton transfers.

Materials and Methods


Site-Specific Mutagenesis

The coding sequence of CA V was derived from BALB/C mouse liver mRNA by reverse transcription and PCR (Heck et al., 1994; Heck et al., 1996). The mutant forms of MCA V used in this study were prepared by Dr. Minzhang Qian in the laboratory of Dr. Philip J. Laipis, and were created using a mutating oligonucleotide (Kunkel, 1985) in the pET31








54











Tyr 64 1







i Lys 170, ...





















Figure 3-1. The location of ionizable residues near the active site cavity of murine carbonic anhydrase V from the crystal structure of Boriack-Sjodin et al. (1995). The three ligands of the zinc are His 94, 96, and 119.








55

expression vector system (Tanhauser et al., 1992); alterations were verified by DNA sequencing.

Expression and Purification

Wild-type and mutant forms of the enzyme were expressed from the pET vector after transformation into E. coli BL21(DE3)pLysS (Studier et al., 1990). All of the expressed enzymes were truncated forms lacking the first 51 amino-terminal residues. In a sequence numbering scheme consistent with CA II, the expressed MCA V variants began at residue 22, Ser. This truncated form of MCA V (denoted MCA Vc by Heck et al. (1994)) has been shown to have identical catalytic properties to MCA V expressed from both a full length coding sequence and a 30 residue truncation of MCA V (Heck et al., 1994).

Purification was performed through previously described procedures with slight modifications (Heck et al., 1994). Frozen cells containing expressed recombinant MCA V mutants were thawed in a solution of 25 mM Tris pH 8.5 containing 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 0.4 mM MgC2, 0.4 mM ZnSO4, 0.1% Pmercaptoethanol, 0.1 mg/mL deoxyribonuclease I and 0.5 mg/mL lysozyme with stirring for two hours in 4C. After cell lysis the cell debris was pelleted by centrifugation at 43000 x g for 15 minutes at 4C. The supernatant was added to a gel filtration column (Ultrogel AcA 44, LKB). The protein eluate was then subjected to affinity chromatography on a gel containing p-aminomethylbenzenesulfonamide coupled to agarose beads as described by Khalifah et al. (1977). Enzyme purity was determined as in Chapter 2 (page 26). Active MCA V mutant enzyme concentration was determined by inhibitor titration of the active site with ethoxzolamide by measuring "0 exchange between CO2 and water (see below). The enzyme was then stored at 40 C.








56

Stopped-flow Spectrophotometry

Initial velocities were determined by following the change in absorbance of a pH indicator (Khalifah, 1971) at 25 C using a stopped-flow spectrophotometer (Applied Photophysics Model SF. 17MV). This method is described in detail in Chapter 2 (page 26) and was used with only minor variations. Here, the CO2 concentrations for the substrates ranged from 0.7 to 17 mM. The buffer-indicator pairs, pK,. values and the wavelengths observed are also described in Chapter 2 (page 26) with two additions as follows: 1,2 dimethyl imidazole (pK = 8.2) and m-cresol purple (pK = 8.3) 578 nm and Ted (pK, = 9.2) and thymol blue (pK, = 8.9) 596 nm.
"0 Exchange

An Extrel EXM-200 mass spectrometer utilizing a membrane permeable to gases was used to measure the rate of exchange of "80 between species of CO2 and water catalyzed by the carbonic anhydrases (equations 2-1 and 2-2, Chapter 2, page 27; Silverman, 1982). Experiments were at 25 C. No buffers were added except where indicated, and a total ionic strength of 0.2 M was maintained with Na2SO4. Solutions contained 25 mM total substrate ([COJ + [HCO3]). This method is described in detail in Chapter 2 on pages 27 through 29. Values of L./K obtained from "O-exchange techniques for the mutants of Table 3-1 were determined by Dr. C. K. Tu (University of Florida). Solvent hydrogen isotope effect experiments are as described on page 29 in Chapter 2.

Results


The mutants constructed for this study have the potential proton transfer residues at positions 64, 91, 131, 132 and 170 replaced with alanine in single and multiple mutations












Table 3-1: Maximal Values of ku/Km and ko,, for CO2 Hydration and kB with pK, Values Obtained from their pH Profiles for Wild-type and Mutants of Murine Carbonic Anhydrase V.
klfK(pM' s") pKa(kcaVt/Km) k. (ms") pK(kt) kB (ms"') pKkB)
Y64A 19 lb 7.8 0.1b 250 50b 9.2 0.lb 80 + 10 8.7 0.3
Y64A/YI31A 22 lb 7.4 0.lb 80 + 0b 8.8 0.lb 30 + 10 > 8.7
Y64A/K91A/YI31A 21 1 7.6 0.1 70 10 9.1 0.1 20 + 10 > 8.5

wild-type 35 la' 7.4 0.1' 320 30a 9.2 0.23 120 20 8.6 0.1
K91A 34 1 7.5 0.1 130 10 9.0 + 0.1 30 10 8.9 0.2
YI31A 37 lb 7.1 0.1b 180 20b 9.2 0.1b 110 + 10 >8.5
K91A/Y131A 36 16' 7.9 0.4 150 30 9.1 0.2 70 + 10 8.3 + 0.2

18 17' 6.9 0.4

K132A 55 1 7.2 0.1 270 20 9.1 0.1 110 20 8.3 0.1
K170A 56 2 7.3 0.1 270 +20 8.9 0.1 140 130 >8.5
Y64A/K91A/YI31A/KI32A 60 2 7.6 0.1 32 6 9.1 0.1 60 10 8.5 0.2
Note: Maximal values of kL,/Km and k were determined from pH profiles for the exchange of "0 measured by mass spectrometry and the maximal values of k, were determined from pH profiles of the hydration of CO2 obtained by stopped-flow spectrophotometry. Heck et al. (1994).
b Heck et al. (1996).
'These standard errors representing the fits to ionization curves are larger than for the other data due to the similar values of the apparent pK...







58


108



107



106








104
5 6 7 8 9
pH










Figure 3-2. The pH dependence of k/Km for hydration of CO2 determined by "0 exchange catalyzed by (0) wild-type MCA V (Heck et al., 1994); (0) K91AY13 1A MCA V; and (A) Y64A/K91AY131A MCA V at 25 0C. Total ionic strength was maintained at 0.2 M by addition of Na2SO4; no buffers were used. Lines are a nonlinear least-squares fit to a single ionization for wild-type and Y64A/K91 A/Y 131 A and to two ionizations for the K9 I A/Y 131 A mutant resulting in the parameters given in Table 3-1.








59

(Figure 3-1, Table 3-1). The pH profiles for k./K. for hydration of CO2, determined from "O-exchange rates, varied as if dependent on the base form of a single ionizable group (Figure 3-2) with apparent values of pK. that were similar for each mutant, from pK 7.1 to 7.9 (Table 3-1). The double mutant K91A/YI31A MCA V was an exception in that two ionizations fit the data better than one; however, the apparent values of pK were very similar for the two ionizations (Table 3-1). The alanine replacements did not cause large changes in the maximal values of /Km which had magnitudes ranging from 1.9 to 6.0 x 107 M's' for all of the mutants studied (Table 3-1). The magnitudes of k./K. appeared to occur in three groups as shown in Table 3-1; for example, the mutants containing Tyr 64 including wild-type MCA V have values of kci/Km very near 3.5 x 10' Ms"' and the mutants containing Ala 64 have values very near 2.0 x 10' M's"' (Table 3-1). The ratio kn/K. contains rate constants for the conversion of CO2 into HC03 up to and including the first irreversible step, the departure of HC03 (equation 1-1, Chapter 1, page 9). Therefore, these mutants are not causing significant changes in this stage of catalysis or influencing the surrounding environment of the zinc-bound water in a manner that would alter its pK, or catalytic activity.

Measurement by stopped-flow of the steady-state constants k., for CO2 hydration had a pH dependence that could also be fit to a single ionization with apparent v alues of pKa in the narrow range of 8.8 to 9.2 for wild-type and mutants (Table 3-1) with typical data shown in Figure 3-3. The variation in the maximal values of k., ranged from 3.2 x l0' s-' for the wild-type enzyme to 0.32 x 10' s' for the multiple mutant Y64A/K91A/Y131A/K132A, which is 10% of k., for the wild-type (Table 3-1).

The 1O-exchange rate constant RH2o/[E] describes the rate of release of H210 from the enzyme into solvent water at chemical equilibrium and involves proton transfer to the






60

106




104

!r 100
j



A

102,,
6 7 8 9 10
pH












Figure 3-3. The pH dependence of k1,, for hydration of CO2 determined by stopped-flow spectrophotometry catalyzed by (0) wild-type (Heck et al., (1994)); (0) K9 1 A/YI 31 A MCA V; and (A) Y64A/K91AYI3 1A MCA V at 25 *1C. Total ionic strength was maintained at 0.2 M by addition ofNa2SO. Lines are a nonlinear least-squares fit to a single ionization resulting in the parameters given in Table 3-1.








61

zinc-bound hydroxide in the dehydration direction (equation 2-1, Chapter 2, page 27). The pH profiles of Rmo/[E] were typically bell-shaped for the mutants of Table 3-1 and yielded a rate constant kB for intramolecular proton transfer and values of pK, for the zinc-bound water and for the intramolecular proton donor determined by least-squares fitting of equation 2-4 (Chapter 2, page 29) to the pH profiles of Ruo/[E]. The values of the pK, of the zincbound water were in agreement, within 0.1 or 0.2 units, with those obtained from the pH profiles of k/Km. Application of these procedures to typical data are shown in Figure 3-4 for wild-type MCA V and K91 A/Y1 31 A MCA V. The '0-exchange results again show a narrow range of values for the proton donor of pK(, from 8.3 to 8.9 (Table 3-1). These values confirm the pK near 9 found from the pH profile of k,, for the proton shuttle also shown in Table 31. The wild-type enzyme had the largest value of the rate constant kB for intramolecular proton transfer with the smallest value being observed for the triple mutant Y64A/K91A/Y13IA at 17% of the wild-type (Table 3-1). The magnitudes of kB and k,. can only be compared qualitatively since they represent proton-transfer in the dehydration and hydration directions.

The solvent hydrogen isotope effect (SHIE) observed for catalysis of CO2 hydration by Y64A/K91AIYl31A MCA V was 1.4 0.2 for kcaK at pH 9.2. This is consistent with no rate-contributing proton transfer in the interconversion of CO2 and HC03" (equation 1-1, Chapter 1, page 9). The SHIE at pH 9.2 on kc,, was 4.1 0.5, consistent with ratedetermining proton transfer involving the aqueous ligand of the zinc (equation 1-2, Chapter 1, page 9).

The capacity of mutants of MCA V to be enhanced in catalysis by proton donors from solution through chemical rescue was also measured by "80 exchange for the







62






0
0 0




100




104




5 6 7 8 9
pH











Figure 3-4. The pH dependence of Rmo/[E], the rate constant for release of "0-labeled water from the enzyme, catalyzed by (@) wild-type MCA V and (0) the mutant K91AYI31A MCA V at 25 0C. The total concentration of all species of CO2 was 25 mM, the total ionic strength of solution was maintained at 0.2 M by addition of Na2SO4, and no buffers were added.








63

dehydration direction of catalysis. These experiments were done using the buffers of small size, imidazole and 1,2-dimethyl imidazole. It was found that imidazole was able to activate RH2o/[E] catalyzed by Y64A/F65A MCA V in a saturable manner at pH 6.3 (Figure 3-5). Imidazole had a slight inhibitory effect on R,/[E] and caused these values to decrease with an apparent K, listed in the legend to Figure 3-5. The maximal value of R1o/[E] when corrected for this inhibition was identical to the value for Y64H/F65A MCA V in the absence of buffer at pH 6.3 (Table 3-2; Figure 3-5). Similar results were obtained through chemical rescue of the triple mutant, Y64A/K91A/YI3 IA, with 1,2-dimethyl imidazole. RI2o/[E] catalyzed by this triple mutant increased in a saturable manner upon addition of 1,2-dimethyl imidazole at pH 8.2 (Table 3-2). The maximal value of RH2/[E] is very similar to that measured in the absence of buffers with Y64H/F65A MCA V at the same pH (Table 3-2). As a control, 1,2dimethyl imidazole catalyzed by Y64A/K91A/YI31A caused no change in R,/[E] at concentrations up to 200 mM.

The capacity of mutants of MCA V to be enhanced in catalysis by proton acceptors from solution through chemical rescue was also measured under steady-state conditions. The estimated values of k,. for CO2 hydration catalyzed by Y64A/K91A/YI31A MCA V increased from a value close to I x 10' s' to values approaching (1.0 0.3) x 10' s- as concentrations of 1,2-dimethyl imidazole increased from I to 200 mM (pH 8.2, 25 C, ionic strength maintained at a minimum of 0.2 M; Data not shown). Again, this is close to the value of kcat (2.0 0.4) x 105 s-' under these conditions for Y64H/F65A MCA V (Heck et al., 1996).

Chemical rescue experiments measured at chemical equilibrium by the "0-exchange technique requires buffers of small size to fit in the active site and directly shuttle protons to







64
100





104









1031

0 50 100 150 200

[Imidazole] mM











Figure 3-5. The dependence of RHo/[E] (O) and R,1[E] (0) as a function of the concentration of imidazole at pH 6.3 and 25 C. The total concentration of all species of CO2 was 25 m.M and the total ionic strength of solution was maintained at a minimum of 0.2 M by addition ofNa2SO4. The data for Rmo/[E] approach a maximal value of(1. I + 0.2) x 10' s"- when corrected for the apparent inhibition manifested in R1/[E] (K, of 0.40 0.17 M). The apparent K. for this buffer was near 91 mM.








65











Table 3-2: Catalysis Enhanced by Proton Donors from Solution in Mutants of MCA V: Values of RH20/[E], the Rate of Release of "O0-labeled Water from the Active Site.

Mutant pH R20/[E]
(x 10' S1)

Y64H/F65A' 6.3 1
8.2 0.2

Y64A/F65A with
imidazoleb 6.3 1.1 0.2

Y64A/K91 A/Y 131 A
with 1,2 dimethyl imidazoleb 8.2 0.2 0.3

Note: All data obtained at 25 0C with a minimum ionic strength maintained at 0.2 M with the addition of Na2SO4.
SC. K. Tu and D. N. Silverman, unpublished data. b Maximal values of R2/[E] were determined from the dependence of RH2/[E] as a function of the concentration of buffer in catalysis by MCA V Y64A/F65A at pH values equal to pK, and under the experimental conditions described in the legend of Figure 3-5. The data for Rmo/[E]I were corrected for the apparent inhibition manifested in RJ/[E].








66

the zinc-bound hydroxide (Tu et al., 1990). Under these conditions, the bulky sized buffer Ted caused no change in RH~J[E] catalyzed by the triple mutant Y64A/K91 AN 13 1 A (at pH 9.3 and 25 'C) up to concentrations of 100 mM. As a control, Ted was also found to cause no change in "J~ catalyzed by Y64A/K91I ANY 13 1 A MCA V up to similar concentrations. This result indicates that Ted cannot directly donate protons to the zinc-bound hydroxide and therefore, does not fit in the active site. However, under steady state conditions where buffer in solution is the final proton acceptor in each cycle of catalysis, chemical rescue of the triple mutant Y64A/K91 ANY13 1lA MCA V was achieved by addition of similar concentrations of the buffer Ted as used in the '80-exchange experiments. In these experiments, Ted produced an increase of greater than 1 0-fold in the initial velocity of C02 hydration. As will be discussed, the comparison of the results of chemical rescue experiments from '0-exchange and steady state techniques provides evidence that the proton transfer groups are located near the surface of MCA V and not buried in the active site.

Discussion


The aim of this study is to identify in MCA V the residue or residues of pK. near 9 that act as proton acceptors in the hydration direction of catalysis. For this purpose the proton transfer capacity was investigated for a number of lysine and tyrosine residues in the active-site cavity and near its rim. Five potential proton shuttle residues in the active-site cavity or near its mouth in MCA V were replaced; this includes many evident basic groups in the vicinity of the active site that could participate as proton shuttles. These have been replaced by alanine and the effect on k. for hydration and on ":0 exchange have been measured.








67

Among the variants of MCA V in Table 3 -1, wild-type MCA V has the largest value of k.~ and the various single mutants with potential proton shuttles replaced have lower values. A similar observation is also made for kB, a rate constant for intramolecular proton transfer determined from "0O exchange, in which again the wild-type enzyme has the largest value (Table 3-1). These decreases in k,,a, for hydration and kB observed in our mutants of MCA V are interpreted as decreased efficiency of the intramolecular proton transfer processes. This interpretation is supported by the following considerations. A wide body of previous data indicates that k., for catalysis by carbonic anhydrase is dominated by intramolecular proton transfer between the zinc-bound water and proton shuttle residues (Steiner et al., 1975; Tu et al., 1989a; Lindskog, 1997), and the studies with murine CA V are also consistent with such rate-determining steps for kcat (Heck et al., 1994; Heck et al., 1996). Many of the previous experiments to confirm rate-limiting proton transfer in this catalysis have been repeated in this study with one of the least active mutants and other mutants of Table 3-1: Y64AIK9IAIYI3 IA has a solvent hydrogen isotope effect of 4.1 on k.a for hydration; kca. is activated by proton donors in solution, an example of chemical rescue; and k,. and RH2/[EI have pH profiles that are consistent with intramolecular proton transfer.

There is no single replacement in Table 3-1 that causes a decrease in k,,, or kB as large as the 40-fold decrease that resulted from the replacement of His 64 by Ala in HCA II (Tu et al., 1989a). Thus, it appears that in MCA V there is not one predominant proton shuttle group as in HCA II but many shuttle groups. The closest potential side chain among those studied is Tyr 64 with its hydroxyl oxygen 7.7 A from the zinc. However, this side chain is








68

pointing away from the zinc in the crystal structure and appears limited in its mobility by the adjacent Phe 65 which most likely accounts for the very slight reduction (or no change, Table 3-1) when it is replaced by alanine (Heck et al., 1994; Heck et al., 1996). Among the remaining lysines and tyrosines of Table 3-1, the closest to the zinc are Tyr 131 with its hydroxyl oxygen 9 A from the zinc and Lys 91 with its NC 14 A from the zinc (BoriackSjodin et al., 1995). Accordingly, these replacements among the single mutants caused the largest decreases in k., (Table 3-1). The residues Lys 132 and Lys 170 have their NC a distance of 19 A and 20 A respectively from the zinc; the decreases in k'.. when these residues are replaced by Ala are very small (or no change) compared with k., for wild-type (Table 31).

The rate constants for intramolecular proton transfer k~ determined by "0 exchange measure proton transfer in the dehydration direction and are different from the values of k, for hydration discussed above. The values of kB add an important component to interpretations of this work since they represent data taken in the absence of buffer, and unlike k,. measured at steady-state, do not contain the possibility of direct proton transfer between the buffer and the zinc-bound water. The decreases in kB for the mutants of Table 3-1 compared with wild-type in general mirror the decreases in k, However there are notable exceptions such as for Y 13 1 A which has a rather greater effect on k,, than on k8 compared with wild-type (Table 3- 1). On the other hand, K91IA has a greater effect on kB. No additional evidence is available to explain these observations.

Hence, among the replacements of basic groups in Table 3-1 resulting in single mutants, the replacements of Lys 91 and Tyr 131 caused significant decreases in kc. for








69

hydration. This evidence is consistent with proton shuttle roles for Lys 91 and Tyr 13 1 as they participate in the intramnolecular proton transfer steps. There are other explanations for these observations, but they can be considered less likely. For example, it is possible that these residues are not proton shuttles themselves but are residues that contribute to proton transfer by their effects on the formation of hydrogen-bonded water networks in the active-site cavity. The following observations indicate that changes in catalysis by the mutants of Table 3-1 are not significantly affected by any changes in such water structure.

First, the lack of a significant effect of the replacement of the suggested proton transfer residues (K9 1, Y 13 1) on kJ, (al3-)cm retowdtyesggests that the chemistry Of CO2 hydration at the zinc is not affected by replacements at these distant sites, possibly including changes in water structure. However, this approach needs further support since studies of human CA II have shown that the insertion of bulky residues including Phe at position 65 adjacent to the proton shuttle residue His 64 alters water structure in the active site of the crystal structure, an effect which is accompanied by significant decreases in k, with smaller or no changes in k.JKX (Scolnick and Christianson, 1996; Jackman et al., 1996). The crystal structures of MCA V and human CA 11 are very similar with backbone conformations that are superimposable with a rms deviation of 0.93 A (Boriack-Sjodin et al., 1995). When the substitution Phe 65 to Ala is made in MCA V, a decrease in k,, is not observed for the resulting F65A mutant compared with wild-type (Table 1 in reference Heck et al., 1996). This suggests that the proton-transfer dependent values of k,,, for MCA V, presumably involving proton transfer from more distant sites, are not affected by changes in water structure caused by the replacement Phe 65 to Ala.








70

Another observation suggesting that changes in water structure are not significantly involved in the data of Table 3-1 is that chemical rescue of certain of these mutants of MCA V with imidazole or 1,2-dimethyl imidazole activates catalysis to levels found for the mutant Y64H/F65A containing an unhindered imidazole as proton shuttle (Heck et al., 1996). Thus, the mutant Y64A/F65A when enhanced with imidazole achieved values of Rmo/[E] near 1 x 10 s' (Table 3-2; Figure 3-5) identical to that of Y64H/F65A. Similarly, the mutant Y64A/K91A/Y13 1A was activated by 1,2-dimethyl imidazole to levels of Ruo/[E] and k, for hydration similar to that of Y64H/F65A in the absence of this buffer (or at very low buffer concentration). These observations also suggest that substitution of K91 and Y 131 on the periphery of the active-site cavity have no measurable effect on proton transfer and presumably water structure when the proton shuttle is 1,2-dimethyl imidazole.

Although Table 3-1 reports decreased catalysis upon replacement of Tyr 131 with Ala, there is the following evidence that proton transfer to enhance catalysis can occur from position 131. Chemical modification of Y 131 C MCA V with 4-chloromethyl imidazole and 4-bromoethyl imidazole caused up to threefold enhancement of Rmo/[E] at pH < 7 with pH profiles consistent with the presence of a proton donor of pK, near 6 (Earnhardt et al., 1998c). These results indicate that the imidazole group of the chemically modified Cys 131 promotes proton transfer and shows that a proton shuttle at this site can act as a proton donor in catalysis. Attempts to observe an enhancement of catalysis with Y131H were not conclusive.

Experiments were performed to eliminate some additional considerations as contri uting to proton transfer, showing they have no significant effect on k,.. For example,








71

rate constants for the initial velocities of catalysis do not increase with an increase of enzyme concentration (Data not shown). Thus, there is no significant intermolecular proton transfer involving ionizable residues on the surface of other carbonic anhydrase molecules in solution. Such a possibility is unlikely due to the sub-micromolar concentrations of enzyme used in all of our experiments.

The data of Table 3 -1 indicate that K91 and Y 13 1 make substantial contributions to proton transfer during hydration, but their replacement still leaves considerable activity, near 3 x 10' s-' at pH near 9 for the quadruple mutant of Table 3-1, which indicates that there remain other proton shuttle residues. Although this is a high rate of catalysis, it is pertinent that proton transfer to hydroxide in solution could be close to this value; k2[O1T] a (109 M''X 10' M) =104 g' at this pH, where k~ is a roughly estimated diffuision-controlled bimolecular rate constant for hydroxide ion encounter with carbonic anhydrase perhaps similar to that found for cyanide (Prabhananda et al., 1987). Although hydroxide might contribute as a proton acceptor, the observation of a plateau in k, at high pH (Figure 3-3) indicates that hydroxide is not the main proton acceptor at pH up to 9. The results indicate that the predominant proton shuttle residues, but not all of the proton shuttle residues, have been accounted for in MCA V. That the remaining catalytic activity in CO2hydration of our least active mutants of Table 3-1 still have a PKa of k., near 9 indicates that the remaining proton shuttles are likely basic residues such as Tyr 58 or perhaps Lys 133 or even more distant basic groups. These basic groups are likely to have a thermodynamic advantage as proton acceptors compared with histidine residues of expected pK, near 6 or 7; besides, the crystal structure of the truncated form of MCA V used in these studies shows no histidine








72

residues near the mouth of the active-site cavity (Boriack-Sjodin et al., 1995). There is a further argument that the proton acceptors unaccounted for lie on the surface of the enzyme rather than deeper in the active-site cavity. That addition of the buffer Ted caused no enhancement of R~mJ[E] catalyzed by Y64AIK91 ANY13 1lA MCA V indicates that this bulky buffer cannot enter the active-site cavity to transfer a proton to the zinc-bound hydroxide -the catalysis is sustained by the various surrounding proton donors (and water) that are at their equilibrium protonation states in this isotope exchange at chemical equilibrium. However, Ted caused a very large increase in the initial velocity of catalyzed C02 hydration suggesting that it can accept protons from proton shuttle sites closer to or on the surface of the enzyme.

Although in single mutants these sites had small changes compared with wild-type, the multiple mutants Y64AIK9I AND1IIA and Y64AIK9IAIYI 3 lA/K1 32A had values of k,. reduced to 22% and 10%/ of that of wild-type while showing no substantial decrease in kca/K. (Table 3-1). This suggests that most of the significant proton shuttles of MCA V have been accounted for and emphasizes that there is no single prominent shuttle as in HCA II, but that a group of residues near the rim of the active-site cavity each make a relatively small contribution to the proton transfer to solution.

The interaction between Lys 91 and Tyr 131 in the catalytic pathway is clearly not additive as indicated by comparison of k,,,, for these single mutants and the double mutant K91I ANY 13 1 A (Table 3 -1). That is, these residues are not acting independently in their role supporting proton transfer. Rather the double mutant causes no additional decrease in catalysis beyond either of the single mutants. This is a form of antagonism, as described by








73

Mildvan et al. (1992), between two residues that becomes evident upon observing catalysis by the double mutant. Two possible explanations account for this antagonistic effect: 1) The side chains of Lys 91 and Tyr 131 are adjacent to one another at the mouth of the active site cavity (Figure 3-1). These two residues form a proton transfer chain in which both are required sequentially to transfer protons out to solution. 2) The antagonistic effect could be structural in which one residue is restricting the mobility of the second to conformations in which proton transfer occurs.

As anticipated, this effect of basic residues is not specific for MCA V. The pH profiles of k,, for at least five of the seven functional isozymes in the a class, CA 1I, 111, IV, V, and VII, demonstrate a dependence on ionizations at high pH which cannot be attributed to His 64 (Silverman et al., 1998). For human CA II this is demonstrated in H64A (Silverman et al., 1998). In human CA III there is an increment in k.,t of unknown source observed at pH > 8 (see Figure 2 of Jewell et al., (1991)). Murine CA IV wild-type has a pH dependence of k., described by two ionizations, and in the H64A mutant the groups with pKa near 9 remain (see Figure 6 of Hurt et al., (1997)). Finally, as described in Chapter 2, the murine form of isozyme VII has a pH dependence for ka. described by two ionizations, one of which is the histidine at position 64 and the other at higher pH is proposed to be another active site residue(s) ionizing at high pH (Earnhardt et al., 1998a). It is possible to consider this common observation for many isozymes of carbonic anhydrase as due to an accumulation of basic amino acids occurring near the active site cavity in many of these isozymes; for example, CA 11, 111, IV, V and VII all contain a lysine at positions 169/170 as well as other lysines and tyrosines located at the mouth of the active site cavity. Thus, in conclusion, in MCA V and








74
likely in other isozymes of the a class of the carbonic anhydrases there are multiple proton transfers contributing to the overall catalytic efficiency of catalysis.














CHAPTER 4
CATALYSIS BY MURINE CARBONIC ANHYDRASE V IS ENHANCED BY EXTERNAL PROTON DONORS


Introduction


Chapter 3 demonstrates that the catalytic activity of MCA V is supported by a number of ionizable residues of basic pKa that act as proton shuttles. Such residues include Lys 91 and Tyr 131 with their amino and phenolic hydroxyl groups 14.4 A and 9.1 A from the zinc (Boriack-Sjodin et al., 1995) and they account for about half of the catalytic turnover (Chapter 3). This finding of multiple proton transfer residues is in sharp contrast to previous studies of CA II that emphasized a single proton shuttle, His 64, that sustains catalysis with a maximal turnover of 106 S-1 (Silverman and Lindskog, 1988; Tu et al., 1989a).

Placing histidine residues at strategic positions in the active site of carbonic anhydrase results in enhancement of catalytic rates for CO2 hydration, some of which approach that of the fastest carbonic anhydrase, isozyme II. Heck et al. (1996) found that replacement of Tyr 64, an inefficient proton shuttle, with a histidine in MCA V and removing a bulky residue at position 65 enhanced the maximal turnover for CO2 hydration to values up to 10-fold over wild-type and 80-fold at physiological pH. Similar results have been obtained upon the replacements Lys 64 to His (Jewell et al., 1991) and Arg 67 to His (Ren et al., 1995) in the least efficient carbonic anhydrase, isozyme III, and with the replacement Asn 67 to His in isozyme II (Liang et al., 1993).


75








76

The enhancements in catalysis caused by these insertions of histidine residues are observed in the steady state rate constant k, for CO2 hydration. These pH profiles of k. can be described by a single ionization with a pKa near 7, suggesting the presence of the inserted histidine residue. There is a small or no effect on k,,JK. compared with wild-type in these mutants. Studies of pH dependencies and solvent hydrogen isotope effects have shown that these intramolecular proton transfers are rate-determining for maximal velocity and that these rate enhancements are specifically attributed to intramolecular proton transfer steps to His 64, just as in isozyme II (Silverman and Lindskog, 1988; Tu et al., 1989a).

In isozyme II and III small buffers in solution, such as imidazole and derivatives, can act as proton donors and acceptors in the catalysis (Tu et al., 1989a; Tu et al., 1990). This is achieved in the mutant of isozyme II with the replacement of the predominant proton shuttle residue, His 64 to Ala (Tu et al., 1989a). These catalytically relevant buffer enhancements have been observed in steady state experiments and "0 exchange at chemical equilibrium and are saturable and consistent with proton transfer to the zinc-bound hydroxide (Tu et al., 1990, 1983; Paranawithana et al., 1990). The maximal rate constants for ratelimiting proton transfer from these buffers yield magnitudes similar to the amino acid counterpart, histidine. Therefore, the introduction of proton shuttles through site-directed mutagenesis or buffers in solution both are capable of achieving proton transfer rates nearly as rapid as in the most efficient carbonic anhydrase II.

Various imidazole, pyridine, and morpholine buffers are used in this study as proton donors to enhance catalysis by a mutant of MCA V lacking a single predominant proton shuttle. The exchange of "0 between CO, and water was measured by mass spectrometry








77

to determine rate-limiting proton transfer from these buffers to the zinc-bound hydroxide in the dehydration direction of catalysis. The values of pKa of the buffers were ranged from 5.4 to 8.6. Similar to previous intramolecular proton transfer studies in mutants of HCA III containing a histidine as a proton shuttle, the rate constants for proton transfer between the proton donor, buffer in this case, and zinc-bound hydroxide as acceptor are described in a free energy plot. Also similar is a curvature in this plot that is characteristic of fast and efficient proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 0.8 kcal/mol) which is also characteristic of nonenzymic rapid proton transfer between nitrogen and oxygen acids and bases in solution. The Marcus parameters yield an observed overall energy barrier (near 10 kcal/mol), indicating that, similar to intramolecular counterparts in catalysis by carbonic anhydrase, there is a large involvement of energy requiring processes such as solvent reorganization or enzyme conformational change. This buffer enhancement study is interpreted in terms of the intramolecular counterparts in catalysis by isozymes of carbonic anhydrase.

Materials and Methods


Site-Specific Mutagenesis. Protein Expression and Purification

The mutant MCA V Y64A/F65A was prepared by Dr. Minzhang Qian as described in Chapter 3 (page 53). Similar to all the MCA V mutants in my work (see Chapter 3), this mutant is a truncated form lacking the first 51 amino terminal residues and therefore, begins at Ser 22, in a sequence numbering scheme consistent with CA II. The expression and purification of this mutant is described in detail in Chapter 3 (page 55).








78
180 Exchange

An Extrel EXM-200 mass spectrometer utilizing a membrane permeable to gases was used to measure the rate of exchange of "80 between species of CO2 and water catalyzed by MCA V Y64A/F65A (equations 2-1 and 2-2, Chapter 2, page 27; Silverman, 1982). This "O-exchange method is carried out at chemical equilibrium and can therefore be performed without buffers, a specific advantage in this study to determine proton transfer from buffers as proton shuttles. Solutions contained 25 mM total substrate (ICO2] + [HCO3]). Buffers were added only where indicated, and a total ionic strength of 0.2 M was maintained with Na2S04 at 25 C. This method is described in detail in Chapter 2 on pages 27 through 29. Solvent hydrogen isotope effect experiments are as described on page 29 in Chapter 2.

Results


Method 1: Saturation Effect of Buffers on RH2E[_EThe effect of two buffers on R1, the rate of interconversion of CO2 and HCO3", and RH2o, the rate of release of '80-labeled water from the active site in catalysis by MCA V Y64A1F65A are shown in Figure 4-1 for 3,5-dimethyl pyridine and in Chapter 3 Figure 3-5 for imidazole. Similar to other buffers of small size in carbonic anhydrase III, these two buffers enhance RHo/[E] in a saturable manner for the mutant MCA V Y64A/F65A (Figure 4-1 and 3-5; Tu et al., 1990, 1983; Paranawithana et al., 1990). This observed saturation is consistent with proton transfer from the buffers to the zinc-bound hydroxide in catalysis. The addition of each buffer listed in Table 4-1 to Y64A/F65A resulted in similar saturation plots for RHo/[E] as shown for the example in Figure 4-1.








79

105






0






104 '"



10 31 1 1
0 10 20 30 40 50 60 70

[protonated 3,5-Dimethyl pyridine] mM





Figure 4-1. The dependence of RH2/[E] (0) and R/[E] (0) as a function of the concentration of protonated 3,5-dimethyl pyridine at pH 6.3 and 25 *C. The total concentration of all species of CO2 was 25 mM and the total ionic strength of solution was maintained at a minimum of 0.2 M by addition of Na2SO4. Rco/[E] data are fit to equation 4-1 yielding a value for ks" of (1.7 0.1) x 10' s"- represented by the solid line. The dotted line is a fit to the same equation for Rmo/[E] data points when corrected for the apparent inhibition manifested in R/I[E] yielding k," of(2.5 0.1) x 104 s"-' and a K.B. KB is listed in Table 4-1. The data points of R,/[E] were applied to a least-squares fit, represented by the solid line, of catalytic velocity to the expression for competitive inhibition as a function of inhibitor concentration under the conditions that the total substrate concentration ([C02] + [HCO3] = 25 mM) was much less than the apparent binding constant for total substrate, Kfis. The K was determined to be 176 42 mM.












Table 4-1: Constants of Equations 4-1 and 4-2 for the Enhancement by Buffers of Catalysis by Y64A/F65A MCA V. Figure 4-4 Donor pK. (Donor)' A pKab kB (x 10-' s-)c KffB (miM)c (kB)H20/ (kB)D20d
a Morpholine 8.6 -1.5 0.6 + 0.1 16 14 2.4 0.3
b 2-Methyl imidazole 8.1 -1.0 1.3 0.3 105 40 1.7 0.1
c 4(5)-Methyl imidazole 7.7 -0.6 1.8 0.2 32 9 2.4 0.6
d 1-Methyl imidazole 7.3 -0.2 3.7 1.0 110 61 1.8 0.2
e 3,4-Dimethyl pyridine 6.7 0.4 4.5 0.5 24 7 2.0 0.4
f 3,5-Dimethyl pyridine 6.3 0.8 3.5 0.2 20 3 3.4 0.1
g 3-Methyl pyridine 5.9 1.3 5.4 0.6 35 10 2.6 0.3
h Pyridine 5.4 1.7 3.0 + 0.7 2.4 1.1 1.7 0.7
1 2,4-Dimethyl pyridine 6.9 0.2 1.9 0.2d 1.5 0.4
2 2,6-Dimethyl pyridine 6.9 0.2 1.2 0.2 19 16 3.0 + 0.5
3 2,5-Dimethyl pyridine 6.7 0.4 1.4 0.2 15 6 2.5 0.2
4 2-Methyl pyridine 6.1 1.0 2.3 0.2 18 5 2.0 0.1
Imidazole 7.3 -0.2 9.6 2.0 82 41 2.1 0.4
z MCA V Y64H/F65A 6.2 -0.1 3.7 0.7
a pK(donor) is equal to log K, in equation 4-2. pl( values were determined from pH titration of 10 mM solutions of buffers with the ionic strength maintained near 0.2 M with the addition of Na2SO4. Standard errors in pK, were less than 1%. b pK,[zinc-bound water] pKa[donor], the pK, (zinc-bound water) was 7.1 0.2 in MCA V Y64A/F65A in the absence of buffer. 'kB' and K. were determined by Method I from the dependence of RH20/[E] as a function of the concentration of protonated buffer in catalysis by MCA V Y64A/F65A at pH values equal to pK, and under the experimental conditions described in the legend of Figure 4-1. Where applicable the data for RH2/[E] were corrected for the apparent inhibition manifested in R1/[E]. Values of k8,* and pK, for the donor and acceptor were then applied to equation 4-2 to determine the k. values listed for each buffer. d Solvent hydrogen isotope effects. Experimental conditions are described in the legend to Figure 4-1. The data are means and standard errors of 3-4 measurements of RH20/[E] using a saturating concentration of buffer. The values of kB were determined from equation 4-2. CData was obtained from the difference in RH2/[E] determined from a pH profile of MCA V Y64H/F65A (C. K. Tu, unpublished data) and MCA V Y64A/F65A, the experimental conditions are described in the legend of Figure 4-2.
0








81

The data of RIJ[E] for 3,5-dimethyl pyridine and each buffer listed in Table 4-1 are described by equation 4-1, which assumes proton transfer from buffers, designated BH, to the zinc-bound hydroxide (Tu et al., 1990).

Rto/[E] = kBap[BH]/(KfB + [BH]) + Rlo(4-1) Here kaB" is the maximal rate constant for the exchange of "O to water that is enhanced by the buffers as proton donors and Kff is an apparent binding constant of the buffer to the enzyme. [E] and [BH] are the concentrations of total enzyme and total buffer. RH2o is the rate of release of "0 into solvent water at zero concentration of buffer and represents the contribution from the enzyme. The experimental values of RH2o/[E] determined by Method I were fit to equation 4-1 using least-squares methods to determine kBw and KnffB. Listed in Table 4-1 are the values of K.f for each buffer. Method 2: pH Dependence of RH_[gJ

The pH dependence of RH2o/[E] catalyzed by MCA V Y64A/F65A was determined in the absence and presence of saturating concentrations of two buffers, 3,5-dimethyl pyridine and imidazole (Figure 4-2). A plot difference plot of Rmo/[E] for the data in Figure 4-2 is shown in (Figure 4-3). The saturation concentration of each of these buffers was confirmed in the experiments described in Method 1. This analysis is similar to previous pH profiles of RH2o/[E] catalyzed by HCA III and mutants of HCA III in the presence of large concentrations of small buffers (Paranwithana et al., 1990; Tu et al., 1990; Jewell et al., 1991).

The rate constant for proton transfer from the donor group to the zinc-bound hydroxide and corresponding pKa values of the donor and acceptor were determined from








82





AA

104- e/





'2 103








102 ,
5 6 7 8 9

pH










Figure 4-2. Variation with pH of Rmo/[E], the proton-transfer dependent rate constant for the release from the enzyme of 1O-labeled water, catalyzed by MCA V Y64A/F65A in the
(0) absence of buffer and in the presence of(E) 100 mM 3,5-dimethyl pyridine and (A) 100 mM imidazole at 25 C. For the data points of Y64A/F65A, in the absence of buffer, the solid line represents a nonlinear least squares fit of equation 4-2 to the data with the pK, of the donor > 9 and acceptor = 6.9 0.3 and the rate constant for proton transfer, kB = (3.7 0.4) x 10 st. The total ionic strength of solution was maintained at 0.2 M by addition of Na2SO4; the total concentration of all species of CO2 was 25 mM.








83

-,4

IT r


-3
~A


2

A










5 6 7 8 9

pH










Figure 4-3. The difference in RHo/[E], the proton-transfer dependent rate constant for the release from the enzyme of "30-labeled water, between MCA V Y64A/F65A in the absence and presence of (0) 100 mM 3,5-dimethyl pyridine and (A) 100 mM imidazole at 25 *C. Experimental conditions are described in the legend to Figure 4-2. The solid lines are nonlinear least-squares fits of equation 4-2 to the data with values of pK for the proton donor, acceptor and k. given in Table 4-2. The fit to the data points for the difference between Y64A/F65A in the absence and presence of 100 mM 3,5-dimethyl pyridine at pH >
7.0 was not considered.








84

the fit of the data in Figure 4-2 and 4-3 to equation 4-2 (this equation is described in Chapter 2, equation 2-4, page 29)

Rfo/[E] = /(1 + KB/[H+])(1 + [H+]/I2)} (4-2)

In equation 4-2, the value of Rmo/[E] can be interpreted in terms of the rate constant from a predominant donor group to the zinc-bound hydroxide (Silverman et al., 1993), in which kB is the rate constant for proton transfer to the zinc-bound hydroxide, KB is the ionization constant for the donor group and KE is the ionization constant of the zinc-bound water molecule. The values of k,, and pK for the donor and acceptors determined for the difference between Y64A/F65A and Y64A1F65A in the presence of saturating amounts of buffer are given in Table 4-2 and described in more detail in the following sections.

In the absence of buffer, kB determined for Y64A/F65A contains a maximum near 4 x 10 s" at high pH (legend of Figure 4-2). This kB associated with high pH is also found for Y64A/F65A catalyzed in the presence of either 3,5-dimethyl pyridine or imidazole buffer (Figure 4-2). This can be interpreted as the contribution to kB from proton shuttles of high pK, on the enzyme; this topic is thoroughly discussed for catalysis by MCA V and mutants in Chapter 3. However, unique to the pH profiles with saturating amounts of buffer is the appearance of maxima near pH 6 to 7. For example, for the addition of 3,5-dimethyl pyridine to the pH profile of Y64A/F65A a maximum appears at pH 6.5 that was not present in its absence (Figure 4-2). By this argument, the maximum near pH 6.5 in the pH profile Y64A/F65A including 100 mM 3,5-dimethyl pyridine is due to the capacity of 3,5-dimethyl pyridine to act a proton donor. This suggests that the buffer 3,5-dimethyl pyridine is contributing to the rate of proton transfer to the zinc-bound hydroxide. This is supported by








85














Table 4-2: Maximal Values of kB with pK, Obtained from their pH Profiles for the Y64A/F65A Mutant of Murine Carbonic Anhydrase V, in the Absence and Presence of Buffer: Method 2.

pK, pK, kB
Buffer (Donor) (Acceptor) (x 10-' s-1')

Y64A/F65A +
3,5-Dimethyl pyridine 6.2 0.1 6.9 0.1 2.5 0.8

Y64A/F65A +
Imidazole 6.4 0.1 7.5 7.2 1.6

Note: All data were obtained by Method 2 with experimental conditions described in Figure 4-3.








86

the difference between the pH profile for RHo/[E] for MCA V Y64A/F65A in the presence and absence of 100 mM 3,5-dimethyl pyridine shown in Figure 4-3. This difference plot shows the bell-shaped pH dependence consistent with the addition of a single proton shuttle and corresponding to an additional proton transfer capacity (RHo/[E]) of (2.5 0.8) x 10' s' with the presence of 3,5-dimethyl pyridine. Similar results were obtained upon addition of imidazole to Y64A1F65A (Figure 4-2 and 4-3; Table 4-2) pK, of the Donors and Acceptors Listed in Table 4-1

p)K, of the zinc-bound water. ka,/Km contains the rate constants for the steps in equation I-1 (Chapter 1), the interconversion of CO2 and HC03 (Silverman and Lindskog, 1988). The pH dependence of lJK for the hydration of CO2 is dependent on the ionization state of the zinc-bound water and therefore yields its pKa (Simonsson and Lindskog, 1982; Lindskog, 1983). The apparent pKa value of the zinc-bound water in the double mutant Y64A/F65A of MCA V was determined from the pH dependence of kc,/Km obtained from SO-exchange methods (Data not shown). A nonlinear least squares fit was applied to the data of k,, and yielded a maximum at high pH and a single ionization with a pKI( value of 7.1 0.2. This value of pK. for the zinc-bound water agrees within experimental error with that determined by Method 2 for pH profiles of RH2/[E] (Figure 4-2). Rmo/[E] is described by the ionization states of the proton donor and acceptor in the dehydration direction of catalysis and the pK, of the zinc-bound water determined by Method 2 is 6.9 0.3. Therefore, the pK, of the zinc-bound water for each additional experiment upon which buffer is added to enhance catalysis was assigned the value of 7.1 (Table 4-1).








87

As a control, this pKa value was compared to those determined by Method 2 in the experiment shown in Figure 4-3, where, saturating amounts of two buffers, 3,5-dimethyl pyridine and imidazole, were added to MCA V Y64A/F65A and RHo/[E] was determined. In each case, the pKa of the zinc-bound water determined from Rwo/[E] was not greatly changed from that of the mutant MCA V Y64A1F65A determined in the absence of buffer (Table 4-2). This finding validates the assumptions given in Table 4-1 that the pK of the zinc-bound water remains relatively unchanged upon addition of buffer to the enzyme.

pI(3 of the Buffers. The pK. values of the buffers listed in Table 4-1 were determined by pH titration of each buffer in solution (see legend of Table 4-1). As described in the previous section the pH profile of MCA V Y64A/F65A was also determined in the presence of saturating amounts of two buffers, 3,5-dimethyl pyridine and imidazole by Method 2 (Figure 4-2 and 4-3). For 3,5-dimethyl pyridine, the pK, determined in the active site of MCA V Y64A/F65A was found to be nearly identical to that determined from titration of the buffer in solution, within experimental error (Table 4-1 and 4-2). Since there is a similarity of pK. values determined through pH titration of the buffers in solution to the pK values determined in Method 2 in the pH profiles of Rmo/[E] for Y64A/F65A, for each buffer in Table 4-1 the pK, is determined by pH titration in solution4. However, an exception is the buffer imidazole that has its pK. for the donor decreased by 0.8 pH units in measurements of



4The addition of large amounts of buffer to the enzyme may change the charge distribution of the active site. In carbonic anhydrase this is possible because the active site is comprised of hydrophobic and hydrophilic sites. To avoid changes in the electrostatic potential of the active site, we held the ionic strength constant at 0.2 M in our buffer experiments. However, large concentrations of buffer may cause an increase in the ionic strength and thereby changes in pK, of the buffer or zinc-bound water.








88

RH2/[E]; but as will be described in the coming sections imidazole behaves differently than the other buffers tested (Table 4-1 and 4-2).

Determination of kn Values of Table 4-1 by Method I

The values of kB listed for the buffers in Table 4-1 were obtained by applying k5,, that was determined in Method 1, to equation 4-2. kBw represents the maximal rate constant for the exchange of "0 at saturating concentrations of total buffer. To determine the maximal rate constant from a protonated donor to an unprotonated acceptor for the dehydration direction of catalysis the value of k,"P must be corrected for the concentration of protonated buffer and unprotonated enzyme as in equation 4-2. Equation 4-2 describes the pH dependence of RH2/[E], the rate of release of "0-labeled water from the active site, which results in a bell-shaped curve for proton transfer from a proton donor and acceptor of equal pK,. The ionization constant for the zinc-bound water applied to equation 4-2 was 7.1 and the ionization constant of the buffers are listed in Table 4-1.

The values of kB determined from Method 1, the buffer dependence of RMO/[E], in Figure 4-1 for 3,5-dimethyl pyridine, are close to the values of kB determined from Method 2, pH profiles of MCA V Y64A/F65A in the presence of a saturating amounts of buffers (Table 4-2). The small difference in the kB value determined from the two methods for 3,5dimethyl pyridine may be a function of buffer inhibition which is accounted for in Method 1, by direct measure of the inhibition in R, (Figure 4-2). Inhibition by these buffers is then corrected in our calculations of RH2/[E] for Method I only (see legend to Figure 4-1). However this is not taken into account with the data obtained from Method 2, from pH profiles (Figure 4-2 and 4-3), therefore this may result in the values of kB being somewhat




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