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1 STRUCTURE AND CATALYSIS AT THE ACTIVE SITE OF HUMAN CARBONIC ANHYDRASE II By BALENDU SANKARA AVVARU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS F OR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Balendu Sankara Avvaru
3 To my parents, Kalyani Devi Tummalacherla and Rajendra Prasad Avvaru
4 ACKNOWLEDGMENTS I am grateful to my mentors, Robert McKenna and David Si lverman. My conversations with them over the years, have taught me much about science and life. Their temperaments have left an impression on me. I will miss them and the times I have spent in their laboratories. Chingkuang Tu was most helpful in many exp eriments I conducted in my research. My doctoral committee; Arthur Edison, Brian Cain and Gerry Shaw, helped me gain better clarity of thought and expression. Mavis Agbandje McKenna was generous in offering her support and the joint resources of the McKenn a Lab. Thanks to these wonderful scientists. I had a great time collaborating with Daniel Arenas, Chae Un Kim and Scott Busby. I am indebted to them for investing their time and energy in my work. I appreciate all the help that was extended to me by the a dministrative staffs of the IDP, BMB and the International student center. Rahul Avvaru, for watching out for me. He totally rocks! Raj Sangani, Jason Gonos, Fred Kweh, Ignacio Sarria, Larry Tartaglia, Mukundh Balasubramanian, Issac Boss, Baber Asad, Nata sha Moningka, Sophia Achee, Emalick Njie, Kinda Seaton, Rose Mikulski, Akin Dubois, and Dean Gurda for all the crazy parties; my most memorable times in America. Tina Halder, Lakshmanan Govindasamy, Arthur Robbins, Kat Sipple, Jason Wagner, Touley Becker, Robert Ng for being great friends and lab mates.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .............. 4 LIST OF TABLES ................................ ................................ ................................ ......................... 7 LIST OF FIGURES ................................ ................................ ................................ ....................... 8 LIST OF ABBREVIATIONS ................................ ................................ ................................ ...... 10 ABSTRACT ................................ ................................ ................................ ................................ 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ................. 15 History of Carbonic Anhydrases ................................ ................................ ....................... 15 Classification of CAs ................................ ................................ ................................ ........... 16 Human Carbonic Anhydrases (HCAs) ................................ ................................ ............. 17 Structure of HCA II ................................ ................................ ................................ .............. 18 Catalytic Mechanism of CAs ................................ ................................ ............................. 19 Substrate Binding in HCA II Active Site ................................ ................................ ........... 20 Methods of Measuring CA Catalysis ................................ ................................ ................ 21 Metal Substitution of HCA II ................................ ................................ .............................. 21 Reflections on Proton Transfer in HCA II ................................ ................................ ........ 22 2 THE METAL BINDING SITE ................................ ................................ ............................. 25 Introduction ................................ ................................ ................................ .......................... 25 Materials and Methods ................................ ................................ ................................ ....... 28 Expression and Purification of Holo HCA II ................................ ............................. 28 Preparation of Apo HCA II ................................ ................................ .......................... 28 Crystallization and X ray Data Collection of Apo HCA II ................................ ....... 28 Structure Determination of Apo HCA II ................................ ................................ .... 29 Hydrogen/Deuterium Exchange (HDX) ................................ ................................ .... 30 Differential Scanning Calorimetry (DSC) ................................ ................................ 31 Circular Dichroism (CD) ................................ ................................ .............................. 32 Preparation of Co(II) HCA II Crystals ................................ ................................ ....... 32 Optical Measurements of Co(II) HCA II Crystals ................................ .................... 33 X Ray Data Collection of Co(II) HCA II Crystals ................................ ..................... 34 Pr operties of Apo HCA II ................................ ................................ ................................ ... 35 Structure of Apo HCA II ................................ ................................ .............................. 35 B factors of Backbone Atoms ................................ ................................ ..................... 37 Melting Temperature ................................ ................................ ................................ ... 38 Backbone Amide H/D Exchange ................................ ................................ ............... 40 Properties of Co(II) Substituted HCA II ................................ ................................ ........... 41
6 Visible Absorption ................................ ................................ ................................ ........ 41 Active Site Cobalt Coordination ................................ ................................ ................. 42 Discussion ................................ ................................ ................................ ............................ 43 3 THE BINDING OF CO 2 AND CATALYSIS ................................ ................................ ...... 64 Introduction ................................ ................................ ................................ .......................... 64 Materials and Methods ................................ ................................ ................................ ....... 65 CO 2 Pressurization ................................ ................................ ................................ ...... 65 X Ray Diffraction and Data Collection ................................ ................................ ...... 66 Structure Solution and Model Refinement ................................ ............................... 66 The CO 2 Binding Site ................................ ................................ ................................ ......... 67 Comparison of Holo and Apo CO 2 Bound HCA II ................................ .................. 67 Secondary CO 2 Binding Site ................................ ................................ ...................... 68 Water Structure and a Short Hydrogen Bond ................................ ................................ 69 Discussion ................................ ................................ ................................ ............................ 71 4 ROLE OF HYDROPHILIC RESIDUES IN THE EXTENDED ACTIVE SITE ............. 81 Introduction ................................ ................................ ................................ .......................... 81 Methods and Materials ................................ ................................ ................................ ....... 84 Expression and Purification of HCA II Mutants ................................ ....................... 84 Crystallization of N62 Mutants ................................ ................................ ................... 85 Crystallization of Y7I HCA II ................................ ................................ ....................... 85 X ray Diffraction and Refinement ................................ ................................ .............. 86 Oxygen 18 Exchange ................................ ................................ ................................ .. 87 Esterase Activity ................................ ................................ ................................ ........... 89 Differential Scanning Calorimetry (DSC). ................................ ................................ 89 Role of the Water Structure and Orientation of His 64 ................................ .................. 89 Asn62 and Tyr7 Contribute to Facile Proton Transfer ................................ .................. 91 Discussion ................................ ................................ ................................ ............................ 96 5 CONCLUSIONS: STRUCTURE CATALYSIS CORRELATIONS ............................. 122 LIST OF REFERENCES ................................ ................................ ................................ ......... 129 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ..... 144
7 LIST OF TABLES Table page 2 1 Refinement and Final Model Statistics for the Crystallographic Study of Apo HCA II ................................ ................................ ................................ ............................... 51 2 2 Thermodynamics of Unfolding of Apo and Holo HCA II ................................ ......... 52 2 3 Data collection and r efinement statistics of crystal structures of Co(II) HCA II at pH 6.0, 8.5 and 11. ................................ ................................ ................................ .... 53 2 4 Geometries of first shell ligands of cobalt in the Co(II) HCA II crystals at (A) pH 11.0; (B) 8.5; and (C) 6.0 ................................ ................................ ........................ 54 3 1 Data and refinement statistics for CO 2 bound holo and apo HCA II crystal structures ................................ ................................ ................................ ......................... 75 3 2 Distance () geometry of CO 2 for holo and apo HCA II ................................ ........... 76 3 3 Refinement and model statistics for 0.9 HCA II crystal structure ....................... 77 4 1 Crystal structu re data and refinement statistics of four variants of HCA II ......... 104 4 2 Mean B factors ( 2 ) for the ordered side chains. ................................ .................... 105 4 3 Ap parent values of pK a obtained by various kinetic measurements of catalysis by HCA II and mutants. ................................ ................................ ............... 106 4 4 Maximal values of rate constants for hydration of CO 2 ................................ ......... 107 4 5 Maximal Values of Rate Constants for Hydration of CO 2 ................................ ..... 108 4 6 Values of Apparent p K a Obtained by Various Kinetic Measurements of Catalysis by HCA II a nd Mutants ................................ ................................ ............... 109 4 7 Thermodynamics of Unfolding of wt and Y7 variants of HCA II ............................ 110
8 LIST OF FIGURES Figure page 1 1 Multiple sequence alignment of 14 human CA isoforms. ................................ ......... 23 1 2 Cartoon representation of HCA II. ................................ ................................ ............... 24 2 1 Overall structure of holo HCA II. ................................ ................................ ................ 55 2 2 Active site of holo and apo HCA II. ................................ ................................ ............ 56 2 3 Plot of the average residu e B factor s of holo and apo HCA II .............................. 57 2 4 Differential scanning calorimetry of holo and apo HCA II. ................................ ..... 58 2 5 Cartoon and surface renditio ns of crystallographic B factors and differential H/D exchange of holo and apo HCA II. ................................ ................................ ... 59 2 6 The pH profiles for the extinction coefficients of crystals of Co(II) HCA II ............ 60 2 7 640 565 for Co(II) HCA II ................................ ................................ ................................ .............................. 61 2 8 The active sites of Co(II) HCA II soaked at (A) pH 11.0; (B) pH 8.5; and (C) pH 6.0. ................................ ................................ ................................ .............................. 62 2 9 Cobalt ligand geometry of Co(II) HCA II ................................ ................................ .... 63 3 1 HCA II structure. (a) Overall view, A close up stereoview of (b) holo and (c) apo HCA II. ................................ ................................ ................................ ...................... 78 3 2 Second CO 2 binding site. ................................ ................................ .............................. 79 3 3 The ordered water network in the active site of HCA II. 80 4 1 The active site of HCA II ................................ ................................ ............................ 111 4 2 The active site of site specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D HCA II. ................................ ................................ ................................ .......... 112 4 3 Structural superposition of the active site of the site specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D with wild type HCA II. 113 4 4 Crystal structures of the active sites of Y7I HCA II superpos ed with wild type HCA II and Y7F HCA II. ................................ ................................ ............................... 114 4 5 Overall (A) and N terminus (B) of superimposed crystal structures of wild type HCA II and Y7I HCA II. ................................ ................................ ....................... 115
9 4 6 The pH profiles for k cat ex /K eff CO2 for the hydration of CO 2 ................................ ....... 116 4 7 The pH profiles of R H2O /[E], the rate constant for variants of HCA II: ................ 117 4 8 The pH profiles for k cat ex /K eff CO2 (M 1 s 1 ) for the hydration of CO 2 catalyzed by wild type HCA II and Y7I HCA II. ................................ ................................ ............... 118 4 9 The pH profiles for R H2O /[E] (s 1 ) for wild type HCA II and Y7I HCA II ................ 119 4 10 Differential scanning calorimetry profiles for Y7I HCA II, Y7F HCA II and wild type HCA II. ................................ ................................ ................................ ................... 120 4 11 Free energy plot of the logarithm of the rate constant for proton transfer k B (s 1 ................................ ................................ ................................ ......... 121 5 2 Proposed mechanisms of HCA II catalysis; Lipscomb and Lindskog ................. 126 5 1 Active site. Su perposition of unbound holo CO 2 bound holo, and bic arbonate bound T200H HCA II ................................ ................................ ............ 127 5 3 Proposed catalytic mechanism of HCA II. ................................ ................................ 128
10 LIST OF ABBREVIATION S Angstrom A alanine AE anion exchange ACZ acetazolamideatm ACES N (2 Acetamido) 2 aminoetha nesulfonic acid BH + protonated base BSA bovine serum albumin C Celsius CA carbonic anhydrase CAPS N cyclohexyl 3 aminopropanesulfonic acid cm centimeter CO2 carbon dioxide p change in h eat capacity E.coli Eschericia coli EDTA ethylenediaminetetraacet ic acid gm gram G Gibbs free energy H + proton/hydrogen ion HCA II human carbonic anhydrase II HCl hydrochloric acid HCO 3 bicarbonate ion HEPES 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid calorimetric enthalpy
11 IPTG isopropyl D thiogalactop yranoside K rate constant k B rate constant for proton transfer K cat turnover number K cat /K m specificity constant kDa kilo Daltons K m Michaelis Menton constant k cat exch /K eff CO2 specificity constant determined for hydration of CO 2 by CA LB luria broth LB HB low barrier hydrogen bond M molar g microgram l microliter M micromolar mg milligram mm millimeter mM milimolar nm nanometer nM nanomolar 18 O stable isotope of oxygen with atomic mass of 18 OD optical density OH hydroxide ion pAMBS para aminomethl ybenzenesulfonamide PDB Protein Data Bank pH negative log of hydrogen ion concentration
12 p K a acid dissociation constant RMSD root mean square deviation Tm melting te mperature Tris tris(hydroxymethyl)aminomethane Zn zinc
13 Abstract of Disserta tion Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURE AND CATALYSIS AT THE ACTIVE SITE OF HUMAN CARBONIC ANHYDRASE II By Balendu San kara Avvaru December 2010 Chair: Robert McKenna Major: Medical Sciences Biochemistry and Molecular Biology Human carbonic anhydrase II (HCA II) is a monomeric zinc containing metalloenzyme that catalyzes the hydration of CO 2 to form bicarbonate and a pr oton. The removal of the zinc alters the collective electrostatics of the apo HCA II that resulted in changes of thermal unfolding, thermal mobi lity of atoms of the apo HCA II. The visible absorption of crystals of Co(II) substituted human carbonic anhydra se II (Co(II) HCA II) were measured over a pH range of 6.0 to 11.0 giving an estimate of pK a 8.4 for the ionization of the metal bound water in the crystal. This is higher by about 1.2 pK a units than the pK a near 7.2 for Co(II) H CA II in solution. CO 2 pre ssurized, cryo cooled crystals were used to capture the first step of CO 2 hydration catalyzed by HCA II the binding of substrate CO 2 for both the holo and apo enzyme to 1.1 resolution. Until now, the feasibility of such a study was thought to be techni cally too challenging because of the low solubility of CO 2 and the fast turnover to bicarbonate by the enzyme These structures provide insight into the long hypothesized binding of CO 2 in a hydroph obic pocket at the active site.
14 Catalysis by HCA II is lim ited in maximal velocity by proton transfer between His64 and the zinc bound solvent molecule. Asn62 and Tyr7 extends into the active site cavity of HCA II adjacent to His64. We compared several site specif ic mutants of HCA II. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer Replacement of Tyr7 by eight other amino acids had no effect on the interconversion of bicarbonate and C O 2 but caused enhancements in the rate constant of proton transfer by nearly 10 fold. The first eleven residues of the amino terminal chain in Y7I HCA II assumed an alternate conformation compared to the wild type. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.
15 CHAPTER 1 INTRODUCTION History of Carbonic Anhydrases T here were two schools of thought on the transport of carbon dioxide in blood prio r to the discovery of carbonic anh ydrase (CA). One pro posed that the hydroph obic carbon dioxide molecule be transported through specialized carrier proteins in blood, which upon reaching the lungs dissociate allow ing the carbon dioxide to be expelled ( 1 ) The second theory put forth the notion that carbon dioxide is likely to be transported in blood as bic arbonate ions. The bicarbonate that is carried to the lungs would be converted back to carbon dioxide by blood proteins and expelled ( 2 ) Thiel conducted early experiments on the uncat a lyzed rate of carbo n dioxide hydration a century ago ( 3 ) He found the rate of hydration to be a meager value of 0.2 s 1 It was clear that this rate was insufficient as the red blood cells have about a second in the lungs to exchange and Henriques using data reported by other r esearchers determined the rate of carbon dioxide hydration under physiological conditions ( 4 ) A catalyst in b lood that speeds up the interconversion of carbon dioxide and bicarbonate was strongly inferred. A few years later, Meldrum and Roughton purified a protei n from ox blood that catalyzed carbon dioxide hydration ( 5 ) And so, the grand story of carbonic anhydrases had begun and it continues to unfold to this day. Carbonic anydrase s since have contributed much to humanity and nearly a century later, they still fascinate scientists from diverse backgrounds.
16 Classification of CAs The carbonic anhydrases found in our biosphere are classified in five evolutionary distinct classes of C As: discovered and placed in the class, although some CAs from mosquito and plant green algae, Chlamydomonas reinhardti have also been grouped in this class ( 6 ) The plant CAs are grouped in the class, with a few exceptions from certain species of bacteria ( 6 ) The CAs from archaebacteria are grouped in the class ( 6 ) classes have r ecently been coined and classified CAs from diatoms and cyanobacteria, respectively ( 7, 8 ) There are many c rystal structures reported for many CAs from and classes (www.pdb.org). To date, there are about 250 CA crystal structures. Most of the deposited human CA isozyme structures are either mutant variants or complexes of the enzyme with inhibitors and/or activators. Liljas et al was the fir st group to determine the CA structure of isozyme II in 1972 ( 9 ) The class CAs were found to be mostly monomers ( 10 ) The crystal structures of and classes followed and were observed in a oligomeric state of dimers and trimers respectively ( 11, 12 ) Although these Zn metallo enzymes are from distinct evolutionary classes and differ in their overall protein folds, they are surprisingly similar in the architecture of their active sites, especially in the vicinity of the Zn b indin g site. CAs are found in many tissue types and draw under their functional significance umbrella, many physiological processes Such as the interconversion of CO 2 /HCO 3 in red blood cells during respiration, acid/base homeostasis, tumor metastasis, bo ne resorption, gluconeogenesis, renal acidification, formation of gastric acid, cellular respiration, cerebrospinal fluid and aqueous humour production ( 13 ) and in plants CAs are speculated to be involved in providing inorganic
17 carbon source (CO 2 /HCO 3 ) for P hosphoenolpyruvate carboxylase and RuBisCO (ribulose 1,5 bisphosphate carboxylase/oxygenase) in the cytosol and chloroplasts ( 14 ) Human Carbonic Anhydras es (HCAs) There are fourteen identified isozymes of CA that are expressed in the human body ( HCA I XIV). The human CAs when compared against each other share an overall sequence identity between 21% to 62% (Figure 1 1). The isozymes vary in their catalyt ic activity and subcellular location; cyto solic (HCA I, II, III, and VII); transmembrane/membrane anc hored (HCA IV, IX, XII and XIV); secretory (HCA VI and XI) ; and mitohondrial (HCA VA and VB) ( 13 ) The amino acids that line the active site are mostly conserved, but small differences in active site residues are capable of fine tuning the catalytic efficiencies of isozymes. HCA I is found primarily in red blood cells; HCA II, although abundantly present in red blood cells is also found to be expressed in all tissues of the human body ( 15 ) HCA III is found in adi pose and muscle tissue. Knock out models of HCA III in mice showed no observable changes in phenotype and hence the precise function of HCA III still remains elusive ( 16 ) ( 17 ) HCA IV is express ed in kidney, lung, and the eye ( 13, 16 ) HCA V is a mitochondrial enzym e ( 18 ) HCA VI is a secreted isozyme found in saliva. It is involved in maintaining the pH balance of the mouth ( 19 ) HCA VII cytosolic and is chiefly expressed in the brain, salivary glands and lungs ( 13 ) HCA VIII, X and XI possess a CA domain, but are inactive isozymes that are designat ed as CA Related Proteins, or CA RPs ( 20 23 ) These inactive isozymes have deleterious mutati ons at the Zn binding sites, that renders them incapable of holding the active site Zn ion. It is interesting to note that the expression of human CA RPs in the brain is developmentally controlled ( 24 ) HCA IX is heavily glycosylated extracellular isozyme and was first observed in cancerous cell lines
18 ( 25 ) Its expression in vivo was observed in renal carcinoma and potentially could serve as a cancer biomark er ( 26, 27 ) HCA XII is expressed chiefly in kidney, colon and prostrate cells. It has a transmembrane domain with the CA domain located on the extracellular side ( 28, 29 ) HCA XIII is a cytosolic isozyme identified in thymus, s pleen and colon. This isozyme has yet to be well characterized ( 30 ) HCA XIV is chiefly expressed in heart and kidney ( 31 ) Structure of HCA II The three dimensional architecture of HCA II was revealed for the first time by Liljas et al through crystallographic methods in 1972 ( 9 ) Its approximate dimensions are 5 x 4 x 4 nm 3 The enzyme assumes a globular shape and could be structurally helices (Figure 2 1) sheet are mainly antiparallel. The first four amino aci ds of the N terminus are usually disordered in the crystal lattice and their conformation cannot be deduced. Deletion studies of the N terminus reveal ed that it is important, neither for the proper folding of the enzyme nor for the catalytic activity ( 32, 33 ) The active site cavity can be loosely descr ibed as being conical in shape, 15 deep and tapers into the center of mass of the enzyme at where the Zn active site metal ion is located This centrally located Zn is coordinated by three histidines (H94, H96 and H119) and a fourth ligand being either a water or hydroxide molecule. This places the Zn in a tetrahedral configuration. The first shell ligands of the Zn are in turn in their place through hydrogen bonding interactions with second shell ligands. His94 and His119 interact with side chains of Glu92 and Glu117 respectively. His96 interacts with the carbonyl oxygen of Asn244. Thr199 and Glu106 help orient the Z n solvent ( 34 ) The active site cavity is partitioned into two very different environments (Fig ure 1 2). On one
19 side of the Zn, deep within the active site, lies a cluster of hydrophobic amino acids (namely; Val121, Val143, Leu198, Thr199 CH 3 Val207 and Trp209). Whereas on the other side of the zinc, leading out of the active site to the bulk solv ent, the surface is lined with hydrophilic amino acids (namely; Tyr7, Asn62, His64, Asn67, Thr199 O 1 and Thr200 O 1). Catalytic Mechanism of CAs The enzyme kinetics of CAs have been studied at length for more than three decades. All the class CAs are deemed to share the same overall ping pong catalytic mechanism com posed of two independent stages as shown in equations 1 and 2 ( 32, 35 ) In the hydration direction, the first stage is the conversion of CO 2 into bicarbonate via a nucleophilic attack on CO 2 by the reactive zinc bound hydroxide. The CO 2 binds in the hydrophobic region of the active site. This binding is discussed in detail in chapter 3. The resultant bicarbonate is then displaced from the zinc by a water molecule (Eq. 1 1). The transient Zn HCO 3 intermediate is speculated to assume two different or ientations in the active site based on the Lindskog and the Lipscomb mechanisms ( 36, 37 ) These mechanisms are discussed in chapter 5. H 2 O CO 2 + EZnOH EZnHCO 3 EZnH 2 O + HCO 3 (1 1) The second stage is the t ransfer of a proton from the zinc bound water to bulk solvent to regenerate the zinc bound hydroxide (Eq. 1 2). The proton transfer is the rate limiting step of catalysis and occurs at an order of 10 6 s 1 ( 20, 38, 39 ) Here B is a proton acceptor in solution or a residue of the enzyme itself. EZnH 2 O + B EZnOH + BH + (1 2)
20 The proton is speculated to hop from Zn bound solvent to His64 through a network of ordered hydrogen bonded water molecules, W1, W2, W3a and W3b in the active site ( 40, 41 ) The protonated His64 loses the proton to BH + an exogenous proton acceptor ( 39 ) Deletion of the imidazole side chain in the mutant H64 A diminished the proton transfer rate by about 10 50 fold ( 42 ) The pH profiles of hydration reveal titration curves that place the pK a close to 7.0, displaying maximal activity at high pH ( 39 ) Of all the human isozymes, H CA II is most efficient at 25 C with a k cat of 1.4 x 10 6 s 1 and HCA III is the slowest with a k cat of 8 x 10 3 s 1 ( 20, 21 ) Substrate Binding in HCA II Active Site The precise binding and orientation of the substrate molecule in the active site of HCA II remained elusiv e for many decades. The failure to directly obs erve the substrate molecule was attributed to the l ow solubility of CO 2 and the extremely high turnover rate of HCA II. Researchers have employed competitive inhibitors to elucidate the substrate binding, however these studies have produced conflicting results as binding modes differ based on the chemical nature of the inhibitors ( 43 45 ) The molecular dynamics studies postulated the CO 2 to bind in the hydrophobic region (Val121, Val143, Leu198, and Trp209) in the vicinity of the active site Zn ( 46, 47 ) CO 2 and HCO 3 are weak binders with a K d of ~100 mM ( 48 50 ) The crystal structure of HCA II reveals a water binding region that is hydrogen bonded to the amide nitrogen of Thr199. It is thought that the infusion of substrate into the active site displaces the deep water prior to the nucleophilic attack ( 51 ) Xue et al captured HCO 3 in the active site of T200H HCA II ( 52 ) The enzyme product complex in this mutant displayed longer half life that allowed for the
21 crystallographic observation. A similar observation in native enzyme has not been reported til l date. We have succeeded in experimentally capturing the CO 2 in the hydrophobic region of the active site in the native enzyme ( 53 ) The characterization of the substrate binding and its implications to catalysis are explored further in chapter 3. Methods of Measu ring CA Catalysis Stopped flow spectrophotometry is employed to me asure CA activity at steady state using pH indicators. K cat (turn over number) and K cat /K M (measure of catalytic efficiency), the initial rates of substrate hydration are determined ( 20, 38 ) 18 O exchange mass spe ctrometry at chemical equilibrium is also employed to determine the kinetic rates of CA catalysis. The method utilizes 18 O, 12 C and 13 C labeled bicarbonate that generates at chemical equilibrium, multiple species of CO 2 differing in molecular weight ( 54, 55 ) R 1 is a measure of substrate hydration, while R H2O is a measure of the rate limiting proton transfer step of catalysis. The R H2O curves infer two ionizing groups (Zn bound water and His64) with pK a values near 7 .0 in the intra molecular proton transfer of HCA II ( 32, 38, 39, 42 ) The 18 O exchange method is thoroughly explained in the methods section of chapter 4. Metal S ubstitution of HCA II Understanding metal binding to proteins in aqueous milieu is important to biological chemistry. Metal ions are strongly bound in proteins primarily through Coulombic stabilization of ionic and dipolar species and to a small extent through hydrogen bonding and van der Waal forces. Zn is most commonly bound ion, second only to iron amongst the deposited metallo protein structures in the Protein Data Bank ( www.pdb.org ). Although HCA II is a Zn enzyme it is capable of binding various metal ions in its active site. DiTusa et al reported the changes in molar heat capacity
22 accompanying binding for Zn ( 117 M 1 K 1 ) and other divalent metal ions ( 56 ) Hakansson et al reported crystal structures of Co(II) Cu(II), Ni(II) and Mn(II) substituted HCA II ( 57 ) Co(II) HCA II is the only variant of the enzyme that has appreciable activity comparable to the native form ( 58 ) The outermost shell of Co(II) has an electronic configuration of d 7 with three unpaired electrons in the ground state ( 59 ) This state is retained in Co(II) HCA II and enables the metal center to shift between tetra and penta coordinated states in response to environmental pH ( 59 62 ) The coordination states of Co(II) and corresponding electronic spectra at various pH values are discussed in chapter 2. Reflections on Proton T ransfer in HCA II The proton transfer in HCA II serves as a model for understanding the directed diffusion of protons in more complex biological systems such as oxidative phosphorylation in mitochondria and photo system s I and II of chloroplasts. The Voth group, in collaboration with the McKenna and Silverman groups has conducted multistate empirical valance bond calculations on the energetically favorable paths a proton may assume in HCA II. Their work also elaborated the importance and contribution of the dual conformation of His64 to the proton transfer step ( 63 65 ) However, classical molecular dynamics simulations place more importance on the contribution of collective electrostatics of the active site than on the dual orientation of His64. The precise mechanisms of proton migration in HCA II are s till under review in carbonic anhydrase circles ( 64 81 ) The effects of Tyr7 and Asn62 (flanking amino acids to His64) on His64 orientation and their implications to proton transfer step are d iscussed in chapter 4.
23 Figure 1 1. Multiple sequence alignment of 14 human CA iso forms. Identical amino acids in the alignment are color coded. Figure generated by ClustalW ( www.ebi.ac.uk/clustalw/ ) and Jalview ( www.jalview.org ) algorithms.
24 Figu re 1 2. Cartoon representation of HCA II The active site residues and CO 2 in the binding pocket are represented as sticks. To depict the dual environment of the active site, hydrophobic amino acids are colored green and hydrophilic amino acids are colore d blue. The active site Zn +2 ion is shown as a grey sphere. The ordered active site waters are shown as red spheres and their probable hydrogen bond network as red dashed lines.
25 CHAPTER 2 THE METAL BINDING SITE Introduction The catalytic active site is characterized by a conical cleft that is approximately 15 deep with the zinc residing in the interior. The zinc is tetrahedrally coordinated by three histdine ligands (His94, His96, and His 119) and a bound water/hydroxyl. The zinc ligand distances are a ll ~2.1 including the zinc bound solvent molecule ( 9, 82 ) It has been previously proposed by Cox et al. that there is a hierarchy of zinc ligands in the active site (that function as distinct shells of residu es to stabilize the zinc ion) ( 83 ) The first shell, or direct zinc ligands, are the three histidi ne residues His94, His96, His 119 and a solvent molecule The second shell, or indirect ligands, stabilize the direct ligands and help position t hem for zinc ion coordination. Residue Gln92 stabilizes His94, Glu117 stabilizes His119, and the backbone carbonyl oxygen of Asp 244 stabilizes His96, while residue Thr 199 hydrogen bonds with the zinc bound solvent. Finally a thi rd shell of stabilization was proposed of a cl uster of aromatic residues (Phe93, Phe95, and Trp 97) that anchor the ains His94 and His96 (Figure 2 1 ) ( 83 ) The first half of this study revisits the study of apo HCA II, and examines the effects of the removal of active site zinc on the structure and stability of the enzyme. The second half exploits the properties of Co(II) substituted HCA II to point out the similarity and differences between crystal and solution structures in understanding enzyme properties and catalytic mechanisms ( 84, 85 ) The properties of the zinc in regards to catalysis are well studied given its importance in the catalytic mechanism. However, its influence on the structural stability
26 of the enzyme has not been thoroughly explored. The active site of apo HCA II has hig h affinity for zinc with a value of pK D near 12 ( 86 ) Hkansson et al. have previously reported the structure of apo HCA II to 1.8 resolution for crystals prepared at pH 7.0 ( 51 ) They n oted no significant differences in structure compare d with holo HCA II. In fact, many of the ordered water molecules of the active site cavity were observed in the apo HCA II as well, although the water molecule corresponding to the location of the zinc bound solvent was shifted ~ 0.8 toward the unoccupie d zinc site. Since HCA II is monomeric, it serves as a good model for a structural stability study on the loss of metal from the active site, as the stability of the enzyme is not complicated by multimeric subunit interactions as observed in other protein structures. Furthermore, the apo and holo HCA II crystallize under similar conditions and were isomorphous in their unit cells. The active site zinc can be removed with chelators and replaced with a variety of metal ions (Co, Mn, Ni, Cu, Fe, Cd) ( 87, 88 ) However, the cobalt substituted enzyme (Co(II) HCA II) is the only derivative with catalytic activity comparable to the native zinc containing enzyme ( 89 ) The crystal structur e of Co(II) HCA II shows minimal changes in amino acid backbone conformation as a result of the metal substitution ( 51, 90 ) The cobalt ion, like the zinc, is coordinated by the same three first shell histidine ligands although the number and orienta tion of the solvent ligands are pH dependent. Hakansson et al. previously solved crystal structures of Co(II) HCA II at pH values of 6.0 and 7.8 ( 90 ) They found s ulfate, an inhibitor of CA, from the crystallization precipitant solution was bound to Co(II) in the structure at pH 6.0 displacing the metal bound solvent with geometry about the cobalt approximately penta coordinate, whilst Co(II) at pH 7.8 was reported to be in tetrahedral coordination.
27 The visible absorption spectrum of Co(II) HCA II is very sensitive to pH. Specifically, the spectra fit a two state model in which the low pH and high pH forms are related to changes in coordination about the cobalt and c hanges in the protonation state of the aqueous ligand of the cobalt ( 87, 88 ) The data indicate an equilibrium between high and low pH forms, and the spectral changes parallel changes in catalytic activity ( 89 ) More detailed solution studies show the titration curve is complex consistent with a smaller influence of other ionizable groups near the active site ( 88, 91 ) The visible spectrum of Co(II) CA observed at high pH shows much stronger absorbance at 640 nm 1 cm 1 1 cm 1 ). The optical spectra of inhibited complexes of CA suggest that the high pH form is associated with a near tetrahedral coordination about the cobalt, and the low pH form is a five coordinate structure ( 61 ) The second half of this study take s adva ntage of the visible spectra of Co(II) HCA II to compare in solution and in crystals the ionization state of the active site, that is the pK a of the cobalt bound water. The visible absorption spectra of crystals of Co(II) HCA II as well as the crystal stru ctures at pH values of 6.0, 8.5 and 11.0 are reported. These are compared with solution properties of Co(II) HCA II including catalytic activity and pK a of the catalysis. Whereas many studies in the literature are consistent with a pKa near 7 for the proto lysis of the metal bound water in HCA II ( 39, 88, 89, 92 ) the crystals of Co(II) HCA II show a spectroscopic pK a of 8.4. This difference is attributed an ionic strength effect caused by the presence of a high concentration of citrate ions in forming crystals. Understanding this difference in pK a between crystal and solution forms of carbonic anhydrase has implications in interpreting pH dependent changes in crystal structures of carbonic anhydrase, such as the pH dependent orientation of the proton
28 shuttle residue His64 ( 93, 94 ) and the observation from neutron diffraction of a metal bound water molecule in HCA II ( 95 ) Materials and Methods Expression and Purification of Holo HCA II The plasmid encoding HCA II was transformed into E.coli BL21 cells through standard procedures and the transformed cells we re expressed at 37 C in LB medium containing 100 g/ml ampicillin ( 96 ) Holo HCA II production was induced b y the addition of isopropyl thiogalactoside to a final concentration of 1mM at an O.D 600 of 0.6 AU. The cells were harvested after 4hrs of post induction. The cell pellets were lysed and holo HCA II was purified through affinity chromatography using pAMBS resin as has been described elsewhere ( 97 ) Preparation of Apo HCA II The zinc was chelated through the incubation of the holo HCA II at 20 C in the chelation buffer (100mM pyridine 2,6 dicarboxyl ic acid ; 25 mM MOPS; pH 7.0) for 8 hrs. The enzyme was buffer exchanged against 50mM Tris; pH 7.8 to remove the chelating agent ( 98 ) The loss of enzyme activity was verified through kinetic studies as has been described elsewhere ( 55 ) The enzyme activity was revived through the addition of 1mM ZnCl 2 attributing the loss of activity to the absence of zinc rather than to the denaturation of the enzyme. Crystallization and X ray Data Collection of A po HCA II Crystals of apo HCA II were obtained using the hanging drop vapor diffusion method ( 99 ) 10 l drops of equal amoun ts of protein and precipitant were equilibrated against precipitant solution (1.3 M sodium citrate; 100mM Tris HCl; pH 9.0) at room temperature (~20 C) ( 82 ) A crystal was cryoprotected by quick immersion into 20%
29 glycerol precipitant solution and flash cooled by exposing it to a gaseous stream of nitrogen at 100K. X ray diffraction data were colle cted at the Cornell High Energy detector distance of 65 mm. Indexing, integration, and scali ng were performed using HKL2000 ( 100 ) Structure Determination of A po HCA II The crystal structure of holo HCA II (PDB accession code: 2CBA) ( 51 ) was used to obtain initial phases of the apo structure using SHELXL ( 101 ) The zinc and all solvent molecules were removed to avoid model bias. 5% of the unique reflections were selected randomly and excluded from the refinement data set for the purpose of R free calculations ( 102 ) Structural refinement proceeded using SHELXL initially with data from 20.0 to 2.0 resolution. The protein geometry was defined using the default constrains of conjugate least squares (CGLS) mode in SHELXL. Each round of CGLS comprised of 15 cycles of refinement. 2F o F c and F o F c electron density Fourier difference maps were calculated after each successive round of CGLS and manually inspected the graphics program Coot ( 103 ) for further fine tuning of the model and the inc orporation of solvent molecules. After some initial rounds of CGLS refinement, the resolution was extended to 1.26 and subsequently after several more cycles of refinement and solvent addition the R work and R free converged to 18.0 and 21.5, respectively. Based on the electron density maps, seven amino acids (Asp 34, His 36, Lys 39, Lys 112, Glu 117, Ser 217 and Glu 238) were built with dual conformations. These dual occupancy side chains were incorporated into the model by splitt
30 In the holo HCA II st ructure these residues were Ile22, Asp34, His64, Asp175, Glu187, Ser217, and Ser 220 ( 82 ) It was noted only residues Asp34 and Ser 217 exhibited dual conformers in both apo and holo HCA II. The rationale for these differences in residues could not be explained structurally. The apo HCA II model was furthe r subjected to several cycles of full anisotropic refinement and hydrogen riding which led a convergence of R cryst and R free to 14.0 and 18.7, respectively. The geometry of the final model was checked using the PROCHECK algorithm ( 104 ) The RMSD for bond lengths and bond angles were found to be within accepted limits of 0.004 and 1.4, respectively. It was observed that 88 % of the dihedral angles were in the most favored region while the rest were in the allowed region with the exception of 0.5 % which were in the generously a llowed region. The average B factors for the main and side chain atoms were determined to be 18.6 and 23.1 2 respectively. The refined model included 250 water molecules with an average solvent isotropic B factor of 30.2 2 The geometry and statistics of the final model a re summarized in Tables 2 1 Hydrogen/D euterium Exchange (HDX) The solution phase amide HDX was performed by the Busby laboratory at the Scripps Institute (Florida), on apo and holo HCA II using a fully automated system that is describ ed elsewhere ( 105, 106 ) Briefly, 4mL of a 15mM protein solution containing either apo or holo HCA II (25mM Tris Cl, pH 7.9, 150mM KCl, 2mM DTT) was diluted up to 20mL with D 2 O dilution buffer (25mM Tris Cl, pH 7. 9, 150mM KCl, 2mM DTT) and incubated at 4 o C for the following periods of time: 1, 30, 60, 900, 3600 and 260000 s. Following deuterium on exchange, unwanted forward and back exchange was minimized and the protein was denatured by dilution to 50ml with 1% T FA in 3M urea
31 (held at 1 o C). The protein sample was then passed across an immobilized pepsin column (prepared in house) at 200ul/min (0.1% TFA, 1 o C) and the resulting peptides were trapped onto a C 18 trap column (Microm Bioresources). Peptides were gra dient eluted (4% CH3CN to 40% CH3CN, 0.3% formic acid over 15 min at 2 o C) over a 2.1 mm x 50 mm C 18 reverse phase HPLC column (Hypersil Gold, Thermo Electron) and electrosprayed directly into a linear ion trap mass spectrometer (LTQ, ThermoElectron). Data were processed using in house software ( 24, 25 ) and Microsoft Excel followed by visualization with pyMol (DeLano Scientific, South San Francisco CA). Average percent deuterium incorporation was calculated for 35 regions of the holo and apo HCA II followi ng 1, 30, 60, 900, 3600 and 260000 s of on exchange with deuterium. To determine differences in exchange between apo and holo HCA II, the average percent deuterium values for all 35 regions of apo HCA II from the average percent deuterium values of the s ame 35 regions of the holo H CA II were subtracted Differential Scanning Calorimetry (DSC) DSC experiments were performed using VP DSC calorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of ~0.5 ml. Apo and holo HCA II samples were buffer ed in 50mM Tris respectively. DSC scans were collected from 30 C to 90 C with a scan rate of 90 C/hr. The calorimetric enthalpies of unfolding were calculated by integrating the area under the peaks in the thermograms after adjusting the pre and post transition baselines. The thermograms were fit to a two state reversible unfolding model to obtain The melting temperatures ( T m ) of apo and holo HCA II occurred at c haracteristic mid points on the DSC curves indicating a two state transition. The difference in Gibbs
32 G ) at a given temperature T was therefore calculated using the following equation ( 107 ) G (T) = H m (1 T/ T m ) + C p [( T T m ) T ln( T/ T m )] (2 1 ) H m is the calorimetric enthalpy at T m C p is the observed change in H S ) were calculated for a give n temperature T using the following H (T) = H m + C p ( T T m ) (2 2 ) S (T) = S m + C p ln( T/ T m ) (2 3 ) Circular Dichroism (CD) The CD spectral analysis was performed by Balasubramanian Venkatakrishnan of the McKe nna lab. The CD data was collected on an AVIV 215 CD spectrometer. Samples of apo and holo HCA II were prepared to 0.4 mg/ml concentration in 20mM Tris pH 7.0 and placed in Starna quartz cuvettes (path length = 0.1 cm) for the data acquisition. T he data was collected from 260 200 nm wavelengths from 38 68 C in 2 intervals to determine the temperature range of the protein melting event. The holo HCA II (control) showed the expected melting temperature ( T m ) of 57 2 C (Figure S4A). Whereas, th e apo HCA II, under the same experimental conditions, showed a significantly lower T m of 47 2 C Using several wavelength regions (ranging from 180 260 to 210 260 nm, in increments of 5 nm increase in the shorter wavelength valve) and CD analysis progr am CDNN ( 108 ) Preparation of Co(II) HCA II C rystals Crystals of apo HCA II were obtained through the hanging drop vapor diffusion method. Ten m l drops of 5 m l of protein and 5 m l precipitant were equilibrated against 1
33 ml precipitant solution (1.4 M sodium citrate; 100mM Tris HCl; pH 9.0) at room temperature (~20 C). The apo HCA II crystals were transferred into soaking solutions of cobalt salt (100 mM CoCl 2 ; 1.4 M sodium citrate; 50 mM Tris Cl; 50mM 3 (cyclohexylamino) propanesulfonic acid (CAPS)) with pH values of 6.0, 8.5 and 11.0, respectively. The crystals were incubated for 2 to 3 d to let the Co(II) ion infuse into the active site. Each crystal was then individually sealed in a quartz capillary tube (outer diameter: 1.0 mm; wall thickness: 0.01 mm) with a small quantity of soaking solution at one end to maintain the vapor pressure and prevent crystal dehydration. Optical Measurements of Co(II) HCA II Cry stals The UV/VIS transmittance of crystals in the capillaries was measured by Daniel Arenas of Tanner lab, UF physics department, using a Zeiss MPM 800 microscope photometer at room temperature (RT) using a beam spot of dimensions less than 100 x 2 Due to the refractive index of the round capillary tubes and of the crystals, refraction can change the beam path, resulting in a portion of the beam missing the detector. To minimize this refraction, the tubes were placed such that the surface of the capi llary tube was normal to the beam path. For the analysis, it was important to show that any amount of light lost due to refraction was independent of frequency. First, the transmittance of empty capillary tubes was measured and found to be constant in fre quency (non dispersive). Then the transmittance of crystals contained in capillaries was corrected using the transmittance of the empty capillary tube. These results showed that the transmittance at longer ested that the amount of light lost to refraction was minimal (< 3 %). As a further check, the transmittance of the crystals was measured for different orientations of the crystal and capillary tube, and it was
34 confirmed that the frequencies of the transmi ssion dips (associated with absorption peaks) were independent of the orientation of the capillary in the beam. Therefore it was concluded that the placement of crystals in capillary tubes did not introduce a frequency dependent error and did not affect th e positions and relative intensities of the absorbance peaks. The absorbance (A) is dependent on the thickness ( d ) of the crystals and the absolute extinction coefficie nts of different crystals could not be compared due to the variation in thickness and shapes of the crystals used. Therefore, the reported extinction coefficients are given in arbitrary units. At lower wavelengths, the visible spectra observed were dominat ed by nonspecific loss of transmission due to light scattering. To subtract this background, the absorption data for each crystal were first fitted to a Lorentzian oscillator model for interband transitions ( 109 ) Then, the high frequency 1 ) were subtracted from the original data. X Ray Data C ollection of Co(II) HCA II C rystals X ray diffraction data sets of Co(II) HCA II crystals at pH values 6.0, 8.5 and 11.0 were collected in house at RT using an R AXIS IV ++ image plate system with Osmic mirrors and a Rigaku RU 100 mA. The image plate crystal distance was set at 80 mm. The oscillation steps were 1 with a 7 min ex posure per image. X ray data processing and scaling was performed using HKL2000 ( 110 ) All three data sets were solved and refined using SHELX97 ( 111 ) The data were phased using the wild type HCA II coordinates (PDB accession code: 2CBA) ( 51 ) with all the solvent, and the Zn(II) active site metal ion removed to avoid model bias. Five
35 percent of unique reflections were randomly selected to serve the purpose of R free calculations. The model refinement using SHELXL97 proceeded initially with the data set from 20.0 to 2.0 resolution. The protein model was visualized and refined using the molecular graphics program COOT ( 112 ) Subsequent cycles of refinement included the incorporation of cobalt, solvent and dual conformers of side chains. The protein geometry was defined by the default constraints of conjugate least squares (CGLS) mode in SHELXL97. Each rou nd of CGLS comprised of 15 cycles of refinement. 2F o F c and F o F c maps were manually inspected after each successive round of CGLS for further fine tuning of the model and the incorporation of solvent molecules. Thereafter, the refinement of the structures of Co(II) HCA II was scaled up to respective maximum resolutions. Table 2 1 provides the final data collection and refinement statistics for the structures determined at pH 6.0, 8.5 and 11. The atomic coordinates for Co(II) HCA II at pH 6.0, 8.5 and pH 11 .0 have been deposited with the Protein Data Bank as entries 3KOI, 3KOK, and 3KON, respectively. Properties of Apo HCA II Structure of A po HCA II S ignificant differences in the overall fold between the apo and holo HCA II structures were not observed Ho wever, the residual F o F c electron density Fourier difference map for apo HCA II displayed some weak electron density at the presumed et al. also reported a similar observation and attributed this to the presence of sodium i ons in their buffers ( 51 ) In this study, the buffers also contained sodium ions and the current data are also consistent with this explanation, or possibly a water molecule, which could have filled the void space left by the removal of the zinc. An alternate, more consistent consideration is that this site contained residual
36 zinc from glassware and solutions, despite care in avoiding this contamination, as apo HCA II has an extremely high affinity for zinc ( 86 ) The apo HCA II structure was refined assuming partial zinc occupancy of 10% based on the relative B factor of the holo HCA II structure at 1.2 resolution In retrospect, the crystallization reagents and cryoprotectent solutions ought to have been treated for removal of exogenous zi nc prior to crystallization. However catalytic ac tivity studies using 18 O exchange methods ( 55 ) estimated the solution sample of apo HCA II to contain a near equal zinc contamination of 10% based on relative activity to holo HCA II. The nature of the apo HCA II in both the crystal and solution was therefore assumed to be similar. The ordered water structure i n the apo HCA II as was identical to that of the active site cavity of holo HCA II. This was also reported by Hkansson et al. ( 51 ) In apo HCA II the solvent molecule corresponding to the zinc bound solvent of the holo H CA II was observed to be shifted ~0.8 towards the unoccupied zinc binding site. In the absence of zinc, this shift may be sterically or energetically favorable. However this observation may also be an artifact of the partial occupancy of zinc. The active site water network has been shown to be supported through a network of hydrogen bonding interactions from the zinc bound solvent and the side chains of residues (Thr 7, Asn 62, His 64 and Asn 67) that line the active site ( 82 ) Previous studies have shown that this active site water network is very sensitive to the amino acid side chain interactions and can therefore be readily disturbed upon mutation of one or more of these residues ( 113 ) As the structure of the apo HCA II reveals no change in the positions of the water molecules in the active site, this would indicate that both the zinc and the zinc bound solvent play little role in the formation and/or stability of the solvent network (Figure 2 2).
37 A comparison between the crystallographic B factors (an indication of the thermal mobility of an atom) of the apo and holo HCA II structures, showed, with the exception of W1, that the solvent had simila r thermal parameters The water molecule W1 in holo HCA II is partly stabilized by a hydrogen b ond with the zinc bound solvent. Hence, in the absence of the zinc, it seems a likely explanation, the removal of this interaction would account for the increased thermal motion of W1 observed in apo HCA II structure In the apo HCA II, His 64 appears pred ominantly (solely within experimental ion with the indole ring of Trp 205. In addition, the apo water molecules, termed W4, W5 and W6, previ ously not observed in the holo HCA II (Figure 2 2 ). The water molecule W4 located in the region that would otherwise be occupied by His 64 in an inward conformer (as observed in the structures of holo HCA II) ( 94 ) The other two additional waters, W5 and W6, are within hydrogen bonding distance to the side chain nitrogens of His64 and are bulk solvent waters, outside the active site cavity (Figu re 2 2 ). B factors of Backbone A toms The apo HCA II structure was compared to the recently reported 1.05 resolution structure of holo HCA II (PDB code 2ILI) ( 94 ) An average B factors for each residue, based on the O, N, and C backbone atoms, in apo HCA II was calculated and normalized to the overall average B value of the holo HCA II. The normalized average B factors for each residue of bot h the structures are plotted in Figure 2 3. The regions indicated by the black bars labeled 1, 2 and 3 in Figure 2 3 were those observed to have significantly larger B factors for apo than for holo HCA II.
38 Inspection of the B factors for the apo HCA II showed that the nearly complete absence of the metal was not associated with significant thermal instability for the residues within the active site cleft, with the exception of Asn67 The histidine ligands of the zinc and secondary shell stabilizing amin o acids showed no increase in thermal vibration in apo compared with holo HCA II (Figure 2 3, dark and light green arrows). But this analysis is suggestive that the absence of the zinc effects thermal stability of residues 53 75, 146 185 and 220 240 on th e protein surface. However, the rate of increase in radial thermal variation as a function of distance from the zinc was no greater than that observe d in the holo HCA II It should, however be noted, that crystal contacts may interfere with the B factor a nalysis, and these results may have been dampened by crystal packing events. Although, again this was not apparent when studying those amino acids on the surface of the enzyme that were involved in such contacts. In Figure 2 3, these residues are indicated by tick marks and show mean average B factor of 25.4 2 while the mean average B factor of the remaining residues is 19.5 2 indicating a mionor difference in crystal versus non crystal contacts. Instead in apo HCA II, the removal of the zinc seems to hav e perturbed the stability of the sheet core (residues 50 75), which in turn seems to affect the stability of s heets of the hydrophobic core that is destabilized in the absence of the zinc Melting Temperature The thermal unfolding transitions of apo and holo HCA II were studied by differential scanning calorimetry ( DSC ) A major unfolding transition, the obser ved dominant peak, was seen for both holo and apo HCA II (Figure 2 4). The transitions
39 were recognized as endothermic peaks, that were centered at the T m with the maximum heat capacity ( C p ) occurring at the T m The T m of holo was observed at 590.5 C w hile that of the apo HCA II was found nearly 8 C lower at 510.5 C. These values were ~2 C lower than the CD values (most likely a consequence of different protein concen tration and buffers) The calorimetric ethalphy at T m H m) was calculated by i ntegrating the area under the unfolding peak, normalized to the protein molar concentration that yielded values of 280 kcal mol 1 and 250 kcal mol 1 for apo and holo HCA II, respectively. Although the experimentally observed DSC curves of apo and holo HCA II were fit to a two 2 2), the DSC curve of apo HCA II does not fit to a two state model as precisely as the holo HCA II due to a secondary peak observed at 58 C (Figure 2 4A). How ever, this could be explained by the 10% of the apo HCA II samples that retained zinc despite stringent efforts to remove it. The DSC curve of apo HCA II when resolved into two distinct peaks in Figure 2 4 B reveals the secondary peak (shown as green in Fig ure 2 4B) to coincides with holo HCA II peak and indicates that the secondary peak could be attributed to a 10% zinc contamination in the apo HCA II samples. These results are in agreement with the crystallographic studies showing residual density that cou ld be account for by assuming ~10% zinc occupancy and the kinetic analysis that also agreed with this G at T m in a two state reversible model is zero, a reference temperature of 55 C, between the melting temperatures of apo and holo HCA II was G T=55 C H T=55 C S T=55 C (Table 2 2 ). The width at half peak height of holo was nearly a unit lower than apo HCA II indicating a relatively higher
40 cooperative transition between the native and denatured states i n the holoenzyme. These results were confirmed using circular dichorism spectroscopy as an alternate technique Backbone Amide H/D E xchange To understand the effects of the loss of zinc on the dynamics of HCA II, comprehensive differential, backbone amide HDX experiments were performed on both holo and apo HCA II. Exchange kinetics were measured for 35 regions of the enzyme and for each region, comparisons were made between the exchange kinetics of the holo and apo HCA II (Figure 2 5 ). As was noted in t he crystallographic B factor comparison, little changes in dynamics were observed when comparing holo and apo HCA II in regions containing the zinc binding sites (His94, His96 and His119) or the cluster of hydrophobic amino acids (namely Val121, Val143, L eu198, Thr199, Val207 and Trp209) that comprise the hydrophobic environment of the active site. However, significant differences were observed in peptides 58 66 that contain important hydrophilic residues of the active site cavity such as Asn62 and the p roton shuttling residue His64. In this case, the amide exchange kinetics for this residue were significantly reduced in holo compared with apo HCA II suggesting a loss of stabilization of this region upon loss of zinc. Moreover, significant differences w ere observed in the amide exchange kinetics of two other regions of the enzyme, residues 147 189 and 224 239 (Figures 2 3 and 2 5). Peptides within these regions demonstrated reduced amide exchange kinetics in the holo compared to the apo HCA II suggesti ng an increase in dynamics in these regions of the apo HCA II which is also consistent with the crystallographic B factor analysis. Taken together, the HDX data corroborates the findings that the loss of zinc does not affect the dynamics of the metal
41 bindi ng site or hydrophobic residues that sequester CO 2 in the active site ( 114 ) but does significantly destabilize important hydrophilic regions of the active site containing proton shuttle His 64 as well as helical regions on the outer surface of the enzyme. Properties of Co(II) Substituted HCA II Visible A bsorption Absorption spectra of crystals of Co(II) HCA II are shown in Figure 2 6 The peak at 640 nm is a major pH dependent absorbance for Co(II) substituted CA II. Although there is considerable scatter in these spectra, the absorbance at 5 65 nm appears to correspond to the isosbestic point that is observed at this wavelength in solution phase, having an identical absorbance for a high pH and a low pH form of Co(II) substituted CA II ( 87 89 ) Due to t he differing sizes and volumes of the crystals, a direct comparison between the extinction coefficients was not possible. However, the data could be normalized to the isobestic point at 565 nm (Figure 2 6 ). Figure 2 7 shows that the ratio of extinction co 640 565 could be fitted to a single ionization, for crystalline Co(II) HCA II prepared from precipitant solutions containing 1.4 M citrate, for Co(II) HCA II in solution containing 0.8 M sodium citrate, and for Co(II) HCA II in solution contai ning 20 mM potassium sulfate, but no citrate, as measured by Taylor et al. ( 115 ) Figure 2 7 shows that the spectroscopic pK a of the crystalline enzyme pr epared in 1.4 M citrate (pK a 8.4 0.1) and the solubilized enzyme in 0.8 M citrate (pK a 8.3 0.1) are shifted to a higher value by about 1.2 pK a units compared with this enzyme in solution in the absence of citrate (pK a 7.2 0.1). Many labs have measure d this pK a to be near 7.0 for Co(II) CA II in the absence of citrate and at lower ionic strength ( 87, 115, 116 ) This shift to a more basic pK a in the presence of citrate is not due to a specific
42 binding site for citrate at the metal in the active site of the enzyme, as there is no evidence of this from the crystal structures, and citra te does not inhibit this enzyme. Active Site Cobalt Coordination The crystal structures of Co(II) HCA II at near 1.5 resolution were solved and refined using standard procedures; the final refinement statistics are given in Table 2 3 No significant changes were observed in the protein backbone and side chains due to the substitution of Zn(II) with Co(II) for crystals soaked at pH 6.0, 8.5 and 11.0. The polypeptide backbone of Zn(II) HCA II (PDB id: 2ili ( 94 ) ) and the solved Co(II) HCA II structures were identical, within the resolution limits of the structures, with a less than 0.2 The main ob servation for this study is that there was no evidence of citrate bound in the active site or on the surface of Co(II) HCA II. The proton shuttle residue His64 in Co(II) HCA II was observed in both inward and outward conformations ( 93 ) with near equal population of each for all three pH values. Unlike Zn(II) HCA II for which there appears to be a single solvent ligand over a range of pH ( 94, 117 ) significant changes in the coordination number as a function of pH were observed in the first shell of the metal ion in Co(II) HCA II crystals. The coordination around the cobalt for crystals soaked at pH 11.0 was tetrahedral with three histidine ligands an d one solvent molecule (Figure 2 8A & 2 9A; Table 2 4 ). This coordination was identical to th at of the tetrahedral coordination of zinc in the native enzyme. The Co(II) HCA II at pH 11.0 was also similar to the Zn(II) HCA II in the locations of the second shell ligands (W DW and W1) and the extended hydrogen bonded water network (W2, W3a and W3b) b eyond the second shell (Figure 2 1 & 2 8 ). When the pH of the crystal soaking solution was decreased to 8.5, the coordination around the Co(II) ion appeared to shift from a tetra coordinated to a
43 coordination resembling more a pen ta coordinated species (Fi gure 2 8 B & 2 9 B). The electron density maps revealed first shell solvent ligands that exhibited a volume too large to account for a single solvent molecule but not large enough for two discrete solvent molecules. The structure therefore was refined assumi ng 50% occupancy for the tetra and penta coordinated Co(II) s pecies (Figure 2 8B & 2 9B; Table 2 4B ). The tetra coordinated species is identical to the tetrahedral geometry of the native Zn(II) containing enzyme, while the penta coordinated species is a s qu are pyramidal geometry (Figure 2 9 B). Upon further decreasing the pH to 6.0, the cobalt ion assumed a hexa coordinated ligation (Figure 2 8 C & 2 9 C; Table 2 4 C). The deep water Dw and W1 from the second ligand shell moved into the first ligand shell. Th ese three first ligand solvent molecules along with the histidine residues are best described a s octahedral geometry (Figure 2 8C & 2 9C ). Discussion The catalytic role of the zinc in carbonic anhydrase, in the hydration of CO 2 is to lower the pK a of th e metal bound hydroxide so as to enhance its nucleophilicity as a Lewis acid ( 32, 34 ) The role of zinc in maintaining the stability of HCA II has been less well studied. Emphasized here is the structural similarity of the apo to holo HCA II at a tomic resolution, a continuation of the initial study of Hkansson et al. ( 51 ) An exception is the conformation of the side chain of His 64, the proton shuttle residue ( 42 ) which in the holo HCA II is equally in an inward and outward orientation that is largely independent of pH between 6 and 8 ( 93, 94 ) whereas in the apo HCA II His64 appears predom inantly in the outward orientation (Figure 2 2 ). This is an interesting result considering that the ordered water in the active site cavity appears nearly identical in
44 both the apo and holo HCA II (Figures 2 1 and 2 2) with very similar B factors (Figure 2 3). Therefore the electrostatic influence of the zinc on the orientation of the side chain of His64 may play a significant role in establishing its dual orientation in the holo HCA II. Moreover, this balance between inward and outward orientation of His6 4 supports a role for both of these conformations in the proton transfer mechanism ( 82 ) The wa ter molecule, W4 in Figure 2 2 was observed in a space vacated by the inward orientation of His64 in the apo HCA II. Although the presence of this additional water is observed only in the apo HCA II structure, its presence in the holo HCA II could well be possible. In the catalytically active holo HCA II, the constant inward and outward flickering of His64 would render this water a momentary presence that would elude its detection through crystallographic (time averaged) methods. However in circumspect, it should be noted that the possibility of the electron density of W4 to be an artifact (owing Nevertheless if the speculation of W4 is true, it would be interesting to deter mine if this water molecule plays any significant role in the proton transfer from the zinc H 2 O to His64. Furthermore, the captured outward orientation of His64 has stabilized two additional water molecules, W5 and W6, which allowed their detection throug h crystallographic methods. These stabilized water molecules in vivo may likely be the first candidates of the bulk solvent to receive protons from the active site during catalysis (Figure 2 2 ), although ultimately protons must transfer to the buffer in so lvent to maintain the maximum velocity of catalysis. The observation of nearly identical ordered water structures in apo and holo HCA II confirms the role of residues in the active site cavity in stabilizing this water
45 network, specifically Tyr7, Asn62, and Asn67, among others ( 113 ) That is, the zinc plays very little, if any, role in stabilizing the structure of the active site waters. This study shows there is no significant change in water structure by removing the zi nc; however, replacement of Tyr 7, Asn62, or Asn67 causes large changes in both the structure of the ordered water and the rate of proton transfer in catalysis ( 113 ) Recent work has also established that the zinc in HCA II plays no role in the binding orientation of CO 2 in the active site cavity ( 114 ) This is further evidence of the predominant role of the zinc in H CA II, to activate the zinc bound solvent molecule for nucleophilic addition to CO 2 Clearly the zinc contributes to the thermal stability of the protein. The main unfolding transition is lowered 8 C in apo compared to holo HCA II (Figures 2 4 and Table 2 2 ). Comparing the crystallographic B factors and backbone amide H/D exchange data to examine the source of this destabilization gave results that were consistent with this observation (Figures 2 3 and 2 5). Residues in the vicinity of the zinc were not observably influenced by its removal. Rather, enhanced thermal mobility in the apo HCA II was observed by B factors and H/D in regions of the enzyme near the surface, especially residues 147 189 containing part of an extended platform of the sheet and an helix (Figure 2 5). However it was interesting to note that the enthalpy of unfolding was similar between the apo and holo G T=55 C yields a small value of 12 kcal mol 1 (Table 2 2). It maybe possible that the surface residues of a fo lded enzyme require the attainment of a threshold vibration for the unfolding cascade to be triggered. This threshold seemed to have to been attained at an earlier temperature of the apo HCA II, possibly because the apo already possessed a higher
46 thermal vibration in comparison to holo H CA II initially (Figure 2 5 ). Both apo and holo HCA II exhibited a two state cooperative denaturation process. The holo displayed a sharper peak indicating a higher extent of the cooperative denaturation than the apo HCA II. It should also be noted that regions of the active site cavity associated with catalysis (except His64) did not change upon removal of zinc. This probably reflects the significance of this region in ordered water formation that maybe critical for pro ton transfer. That is, mobility in this region would decrease catalysis. The enhanced mobility in apo HCA II of regions away from the zinc and closer to the surface of the enzyme simply reflect weaker intramolecular interactions in these regions. The sourc e of this effect may again be electrostatic due to removal of the zinc. It may also be due to an angular effect; that is, small mobility near the zinc binding site where there are significant intramolecular interactions may translate to more extensive mobi lity near the surface of the enzyme where there are fewer such interactions. With the accumulation of considerable kinetic and crystallographic information on the carbonic anhydrases, it becomes useful to compare active site properties in solution and in crystalline states. For example, catalysis by HCA II is very pH dependent ( 32 ) ; it is necessary to compare crystal structures and kinetic data under similar conditions. We have approached this by placing cobalt at the active site of HCA II and using its visible spectrum as a reporter both in solution and in crystals. Here we point out a significant difference in the pK a of the cobalt bound water in crystal versus solution phase. The result we emphasize is that the spectroscopic pK a of 8.4 for Co (II) HCA II in the crystal is considerably larger than the often measured pK a near 7.0 in solution (Figure 2 7).
47 This difference in pK a values is possibly a result of the significant differences in ionic strength. In this study, citrate was used in the cr ystallization precipitant solutions because it does not bind in the active site of Co(II) HCA II, unlike sulfate. We know this because no ordered anion is observed in the crystal structures (Figure 2 8), and citrate was shown not to inhibit catalytic acti vity. In contrast, sulfate was avoided in precipitant solutions, as previous studies of HCA II ( 113 ) and Co(II) HCA II ( 90 ) using sulfate resulted in sulfate bound in the active site, which impairs analysis of structure function data and the role of ordered water in the active site cavity. We also attempted to f ind other anions that might be useful in crystallization, however many including malate, oxalate, and glutamate were excluded because they were also all found to be inhibitors of carbonic anhydrase. The precipitant solution in the current studies containe d 1.4 M citrate; this is an ionic strength of 4.8 M. Of course, this does not necessarily reflect the citrate content of the crystals. The visible spectrum of the solution form of Co(II) HCA II in 0.8 M sodium citrate was fit to a pK a of 8.3 (Figure 2 7), nearly identical to the pK a obtained from the crystal. By this measure the crystal behaves as if it has the equivalent of 0.8 M citrate. The solution data (Figure 2 7) show a pK a of 7.2 at an ionic strength of 0.06 M due to potassium sulfate. Despite decad es of study on the carbonic anhydrases, there is very little examination of the influence of ionic strength on the properties of the active site such as the pK a of the aqueous ligand of the metal. This is mainly because of the difficulty in finding anions that do not bind or interact in a manner that perturbs structural or catalytic properties. Jacob et al. ( 118 ) measuring the solvent relaxation of protons of an
48 extensively dialyzed sample of Co(II) bovine CA II, found a pK a as low as 5.2 at very low ionic strength. This pK a was obser ved to be 6.4 in Na 2 SO 4 at an ionic strength of 0.3 M. The effect of sulfate is in part a contribution to the ionic strength of solution and in part specific binding of sulfate at the active site as measured by inhibition of catalysis ( 119 ) However, the data indicate and Jacob et al. conclude that the pK a of the metal bound water is highly dependent on ionic strength. This conclusion was also reached by Pocker and Miao ( 120 ) who determined that the pK a of the zinc bound water in b ovine CA II increased from 5.9 to 7.0 as the ionic strength of solution was raised from zero to 0.1 M using sulfate. We examined the crystal structures of Co(II) HCA II obtained from solutions containing 1.4 M citrate to determine if there are structural changes compared with the many structures reported for HCA II under a variety of conditions. These structures of Co(II) HCA II are similar in many aspects with structures determined previously ( 90, 121 ) The structure at pH 11.0, above the pK a of the metal bound water, showed tetrahedral coordination essentially identical wi th the Zn(II) containing native enzyme ( 34, 94, 117 ) and is associated with the strong absorbance at 640 nm (Figures 2 1 and 2 8A). The crystal structure at pH 8.5 was refined as an intermediate between tetr a and penta coordinated species and its absorbance at 640 nm occupies a position between the strong and weak absorbance of tetra and penta coordinated species respectively (Figure 2 8B). It is interesting to note that the Co(II) HCA II crystals at pH 8.5 (Figure 2 8B) are near the pK a at the active site of the crystalline enzyme and lie at an intermediate position between tetra and penta coordinated species of Co(II) from the standpoint of structure and visible spectra.
49 The crystal structure at pH 6.0 d isplays hexa coordination about the cobalt with metal solvent ligand distances of less than 2.5 (Table 2 4C and Figure 2 9C). The metal ligand distances have relaxed by ~0.3 suggesting a Co(III) state. There is no evidence from previous studies of vis ible spectra or of crystal structures for a hexa coordinated species of Co(II) substituted carbonic anhydrase ( 34, 51, 88, 117 ) On the contrary, the electronic spectra of the oxidized form Co (III) HCA II have been associated with octahedral complexes ( 122, 123 ) In this respect the hexa coordinate structure at pH 6.0 suggests that the cobalt has been oxidized in our experiments; this is not unexpected since the oxidation of Co(II) is more rapid at low pH. Although the distances of the three solven t ligands (CoOH /H 2 O, W1 and W DW ) observed at pH 6.0 were within the first ligand shell, only the CoOH /H 2 O and W1 were strongly bonded to the metal with B factors of 4.2 and 6.5 2 respectively. The deep water W DW appears weakly associated to the metal ex hibiting a B factor of 22.5 2 These results have implications in the interpretation of structures of crystals prepared in solutions containing citrate. For example, in the first structure of a carbonic anhydrase by neutron diffraction, Fisher et al. ( 95 ) have prepared crystals of HCA II in precipitant solutions containing an initial concentration of 1.15 M sodium citrate at pH 9.0. The ionization state of the zinc bound solvent from the neutron diffraction study is more readily understood by assuming that the presence of citrate has increased the pK a by 1.2 units, as observed in Figure 2 7, and by 0.5 to 0.7 units for the in crease in dissociation constant of normal acids when deuterated ( 124 ) This brings the estimated pK a at the metal in HCA II from 7 to near 9 and more readily explains the observed metal bound D 2 O in crystals equilibrated at pH 9.0. The co nclusions of this current
50 study need also be considered in assessing the role of the proton shuttle His64 of HCA II which according to crystal structures has a pH dependent conformational change ( 93, 94, 125 )
51 Table 2 1. Refinement and Final Model Statistics for the Crystallographic Study of Apo HCA II Data collection statistics Space Group P2 1 Unit cell parameters (,) a = 42.7, b = 41.6 c = 72.8, =104.5 Resolution () 50 1.26 (1.31 1.26)* R sym 0.057 (0.194) Completeness (%) Redundancy 29.7 (10.0) Total number of u nique reflections 61768 (5933) a R cryst (%) b R free (%) 14.0 18.7 Residue Nos. 4 261 No. of protein atoms 2087 No. of H 2 O molecules 250 B factors ( 2 ) Aver age, main side chain, solvent 18.6, 23.1, 30.2 Ramachandran statistics (%) Most favored, addi tionally allowed and generously allowed regions 88, 11.5, 0.5 R.m.s.d. for bond lengths (), angles () 0.004, 1.4 *Values in parenthesis re present highest resolution bin a R symm b R cryst F o| | F F obs | ) x 100 c R free is calculated in same manner as b R cryst except that it uses 5% of the reflection data omitted from refinement.
52 Table 2 2. Thermodynamics of Unfolding of Apo and Holo HCA II Par ameter Apo HCA II Holo HCA II T m (C) a 510.5 590.5 H m (kcal mol 1 ) a 28020 25020 H vH (kcal mol 1 ) b 20020 22020 C p (kcal mol 1 K 1 ) c 0.86 0.75 G T=55 C (kcal mol 1 ) d 9.3 +3.0 H T=55 C (kcal mol 1 ) e 283 247 S T=55 C (kcal mol 1 ) f 0.87 0.74 a Calorimetri c parameters determined by DSC b H vH ) was determined by fitting thermograms to a two st ate reversible unfolding model c C p ) of protein unfolding obtained by plotting calorimetric enthalphy H m) vs mel ting temperature ( T m ) d, e and f Thermodynamic parameters extrapolated to reference temperature of 55 C u sing eq 3, 4 and 5 respectively
53 Table 2 3. Data collection and refinement statistics of crystal structures of Co(II) HCA II at pH 6.0, 8.5 and 11. pH 6.0 pH 8.5 pH 11.0 Space Group Unit cell dimensions, a, b, c (), ( ) P2 1 42.8,41.7,72.9,104.6 P2 1 42.6,41.8,72.8,104.6 P2 1 42.7,41.7,72.8,104.6 Resolution () 50 1.6(1.65 1.60) 50 1.5 (1.55 1.50) 50 1.5 (1.55 1.50) R symm a 8.0 (43.0) 16.0 (5.0) 7.4 (44.0) 28.0 (4.0) 6.5 (32.0) 20.0 (6.0) Completeness ( %) 92.4(90.5) 95.8 (91.8) 97.9 (98.1) Redundancy 2.4 (2.0) 3.5 (2.8) 3.3 (3.1) No. of unique reflections 29472 (3021) 36608 (3472) 38235 (3815) R cryst b / R free c 18.9 / 21.5 19.4 / 22.7 20.1 / 23.5 No. of protein/solvent atoms Average B factors( 2 ) main/side chain Co/solvent 2048/138 11.7/17.4 5.0/21.0 2068/223 15.9/21.0 8.8/30.2 2068/212 14.2/18.8 6.6/26.0 Ramachandran statistics (%) F avored, additionally and generously allowed regions 88, 11.5, 0.5 88, 11.5, 0.5 88, 11.5, 0.5 R.m.s. d. for bond lengths (), angles () 0.006 1. 5 0.008 1. 6 0.008 1.4 Values in parentheses refer to the highest resolution bin a R symm b R cryst F o| | F F obs | x 100 c R free is calculated in same manner as R cryst exc ept that it uses 5% of the reflection data omitted from refinement.
54 Table 2 4. Geometries of first shell ligands of cobalt in the Co(II) HCA II crystals at (A) pH 11.0; (B) 8.5; and (C) 6.0 Angle X Co Y ( ) A) pH 11.0 His119 CoOH/H 2 O 112 116 117 104 112 95 B) pH 8.5 Alternate ligand 1 Alternate ligand 2 CoOH/H 2 O 111 118 118 104 111 93 90 93 160 99 89 152 Alternate ligand 1 71 C) pH 6.0 W1 Dw CoOH/H 2 O 94 159 95 82 72 98 110 85 165 97 82 94 165 77 W1 87
55 Figure 2 1 Overall structure of holo HCA II. Cartoon depiction of the seconda ry structural elements are as shown. Str ucture used was (PDB ID: 2ILI ). Zinc atom represented as a grey sphere, histidine residues His 94, His 96, His 119, and the dual conformation of His 64 are represented in stick model representation (carbon, yellow; oxygen, red; nitrogen, blue). Figure made using PyMOL (DeLano Scientific).
56 Figure 2 2. Active site of holo and apo HCA II. Stereoview of the active s ite of (A) holo (PDB ID: 2ILI) and (B) apo HCA II (this study). The amino acids are as labeled and re presented in stick form (carbon, yellow; oxygen, red; nitrogen, blue). All shown electron density for both the holo and apo HCA II is weighted 2 Fo Fc waters are colored red and the additional waters in apo HCA II (W4, W5, and W6) is colored green. Figures are made using PyMOL (DeLano Scientific).
57 Figure 2 3. Plot of the average residue B factors of holo (dark blue) and apo HCA II (pink). Regions labeled 1, 2 and 3 (indicated by the black bars) are segments of apo that have higher B factors than holo HCA II. The green bars at the base of the figure indicate regions that have the largest extent of difference (up to 12%) in H/D exchange rates between apo and holo HCA II. The dark green arrows indica te first shell ligands, whilst light green arrows indicate second shell ligands of the zinc in holo HCA II. The tick marks above the abscissa represents regions of crystal contacts within the crystal lattice.
58 Figure 2 4. Differential scanning calorimet ry of holo and apo HCA II. (A) DSC scans from 30 to 90 C of holo (black) and apo HCA II (red), shown the dominant transition peaks with values of T m of 59 and 51 C respectively. (B) The DSC curve of apo HCA II from panel (A) was resolved into two indep endent peaks red and green. The secondary peak (green) coincides with holo HCA II peak (black) and is attributed to 10% holo HCA II contamination within apo HCA II samples (see text for explanation).
59 Figure 2 5. Cartoon and surface renditions of cryst allographic B factors and differential H/D exchange of holo and apo HCA II. Cartoon (A) holo and (B) apo HCA II color coded based on their respective normalized B factors. The color transition from blue to red depicts the relative increase of B factor f rom the lowest to highest (see Figure 3). (C) Surface profile comparing backbone amide exchange rates of apo HCA II to holo HCA II overlaid onto the apo HCA II crystal structure. Differences in deuterium incorporation for each of 36 regions of HCA II were determined by subtracting the mean exchange rates for each region of the apo HCA II from the mean exchange rates of the same regions of the holo HCA II for the exchange times of 1, 30, 60, 900, 3600 and 260000 s. Regions colored yellow represent increase s of H/D amide exchange rates between 5 9% and regions colored orange represent increases of H/D amide exchange rates between 10 20% of apo HCA II relative to holo HCA II. Regions colored gray represent areas of non significant changes in H/D amide exchang e rates between the two forms of the enzyme and regions colored white could not be experimentally measured. Figures made using PyMOL (Delano Scientific).
60 Figure 2 6 T he pH profiles for the extinction coefficients (a.u. is arbitra ry units) of crystals of Co(II) HCA II (dimensions approximately 0.2 x 0.2 x 0.2 mm 3 ) mounted in quartz capillary tubes (outer diameter 1.0 mm, wall thickness 0.01 mm). The UV/VIS transmittance was measured using a Zeiss MPM 800 microscope photometer at r oom temperature using a beam spo t of 2 The extinction coefficient of each crystal was scaled to be unity at the isosbestic point at 565 nm.
61 Figure 2 7. The pH dependence of the 640 565 for Co(II) HCA II using ( red ) the data of Figure 2 for crystalline enzyme; ( black ) solutions of Co(II) HCA II containing 0.8 M sodium citrate, 25 mM Tris and 25 mM 2 (N cyclohexylamino)ethanesulfonic acid (Ches); and ( blue ) the data of Taylor et al. ( 115 ) for Co(II) HCA II in solution containing 20 mM potassium sulfate. The absorbance at 640 nm is a major, pH dependent band, and the absorbance at approximately 565 nm is an isosbestic poi nt for two forms of Co(II) substituted CA II, one with a water molecule coordinated to the metal and one with hydroxide. The solid lines are fits to a single ionization with values of pK a of 8.4 0.1 for the crystalline enzyme; 8.3 0.1 for solubilized e nzyme in 0.8 M citrate; and 7.2 0.1 for the solubilized enzyme in the absence of citrate.
62 Figure 2 8 The active sites of Co(II) HCA II soaked at (A) pH 11.0; (B) pH 8.5; and (C) pH 6.0. His 64, the proton shuttle residue, was observed in dual inward and outward conformations in the crystal structures. The ordered water structure in the active site cavity (W1, W2, W3a and W3b) was the same as that observed for the Zn(II) containing enzyme. All electron density shown is repre weighted 2 Fo Fc Fourier map. The angles and distances of the first shell cobalt ligation are given in Table 2 and Figure 5. Figure made using PyMOL (DeLano Scientific). A B C
63 Figure 2 9 Cobalt ligand geometry of Co(II) HCA II soak ed at (A) tetrahedral, pH 11.0; (B) pentagonal pH 8.5; and (C) octahedral pH 6.0. Distances () are as depicted. Angles are given in Table 2. Figure made using PyMOL (DeLano Scientific).
64 CHAPTER 3 THE BINDING OF CO 2 AND CATALYSIS Introduction The visual ization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO 2 pressurized of cryo cooled crystals is used to capture the f irst step of CO 2 hydration catalyzed by HCA II the binding of substrate CO 2 for both the holo and apo (without zinc) enzyme to 1.1 resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low s olubility of CO 2 and the fast turnover to bicarbonate by the enzyme ( 46 ) These structures provide insight into the long hypothesized binding of CO 2 in a hydrophobic pocket at the active site, and demonstrate that the zinc does not play a critical role in the binding or orientation of CO 2 This method may also have a much broader implication for the study of other enzymes for which CO 2 is a substrate or product, and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. The active site cavity of HCA II is partitioned into two very different environments. On one side of the zinc, deep within the active site, lies a cluster of hydrophobic amino acids (namely; V121, V143, L198, T199 CH 3 V207 and W209). Whereas on the other side of the zinc, leading out of the active site to the bulk solvent, the surface is lined with hydrophilic amino acids (namely; Y7, N 6 2, H64, N67, T199 1 and T200 1 ) (Fig ure. 3 1 ). Previously, molecular dynamics studies have implied that the hydrophobic region of the active site sequesters the CO 2 substrate and orients the carbon atom in readiness for n ucleophilic attack by the zinc bound hydroxide (Eq. 1 1 ) ( 46, 47 )
65 Additionally, crystallographic studies have identified an ordered water molecule, DW that is stabilized by the amide nitrogen of Thr199 and th e zinc bound hydroxide It has been proposed that this water is likely displaced upon the infusion of CO 2 into the binding pocket ( 47, 126 ) The hydrophilic wall of the active site has been shown, by X ray crystallography, to create a well ordered hydrogen bonded solvent network. It is hypothesized that this network is required to permit the transfer of a proton from the zinc bound water to the bulk solvent via the experimentally identified proton shuttling residue His64 (Eq. 1 2) ( 40, 42, 73, 82 ) Taken together, these two very different active site environments permit the sustained and rapid catalytic cycling of CO 2 to bicarbonate. Materials and Me thods CO 2 Pressurization I n order to capture CO 2 in the active site of HCA II it was essential to cryo cool the crystals under CO 2 pressure. This was achieved using the high pressure cryo cooling method that was originally developed for crystal cryoprote ction ( 127 ) The crystals were first soaked in a cryo solution containing 20% glycerol in precipitant solution The crystals were then coated with mineral oil to prevent crystal dehydration and loaded into the bottom of high pre ssure tubes. In the pressure tubes, the crystals were pressurized with CO 2 gas at 15 atm at room temperature. 25 min later, without releasing CO 2 gas, the crystals were slowly frozen over 2 min by dipping the sealed end of the pressure tubes into liquid ni trogen. During the cooling process, it was noticed that the CO 2 gas pressure gradually dropped from 15 atm to below 1 atm due to CO 2 solidification.
66 X Ray D iffraction and Data C ollection Diffraction data were collected at Cornell High Energy Synchrotron So urce (CHESS), beamline A1 at a wavelength of 0.9772 Data were collected using the oscillation a crystal to detector distance of 65 mm. A total of 624 and 360 images were collected for the holo and apo data, respectively. Indexing, integration, and scaling were performed usin g HKL2000 ( 100 ) The crystals of the CO 2 bound holo and apo HCA II diffracted to 1.1 resolution and were processed to a completeness of 9.9% and an R sym of 8.8%, and completeness of 93.1% and an R sym of 8.0%, respectively. Complete processing statistics are given in Table 3 1. Structur e Solution and Model Refinement T he structures of CO 2 bound holo and apo HCA II were solved in a similar manner using the program SHELXL ( 101 ) Prior to refinement, a random 5% of the data were flagged for R free analysis ( 102 ) The previously determined 1.54 resolution crystal structure of holo HCA II (PDB ID: 2CBA) ( 51 ) was stripped of all waters, the zinc, and any alternate con formers and used as the initial phasing model in a round of least squares, rigid body refinement to 2.5 resolution to an R factor /R free of 31.3/33.2 % for holo and 28.0/28.6 % for apo enzyme. The data was then extended to 1.5 resolution and the model w as refined using conjugant gradient least squares (CGLS) refinement. After 20 cycles, the model and related sigma weighted electron density maps were read into the molecular graphics program Coot ( 103 ) Improperly built side chains an d the zinc (in the holo structure only) were placed into their respective density and the model was run through another round of CGLS refinement. Waters with positive density in the sigma weighted difference map were kept until all waters with reasonable density were
67 built. The data sets were then extended to 1.1 resolution and the final waters were built. Disorder was then modeled into the density by modeling all visible alternate conformations for both amino acid side chains and waters. Riding hydro gens were then placed on all residues except the imidazole nitrogens of the histidines. The weighting factor was then changed to 0.2 for one round followed by the use of all data for the final round. The final R final /R free for holo was 10.9/12.9% and for apo was 10.4/13.9%. Complete refinement statistics can be found in Table 1. The model geometries and statistics were analyzed by PROCHECK ( 104 ) The CO 2 Binding Site Here we describe for the first time, to our knowledge, the experimental capture of CO 2 in the hydrophobic cavit y of HCA II (Fig ure 3 1 ). The holo and apo HCA II CO 2 bound structures were refined to 1.1 resolution with final R factors of 10.90 and 10.35, respectively (Table 3 1). Both exhibited only minor structural perturbations compared to the holo unbound stru cture (PDB ID: 2ILI) (13), with C RMSDs of 0.21 and 0.15 respectively. The active site bound CO 2 molecules for both the holo and apo HCA II structures were clearly seen in the initial F o F c electron density maps, on the hydrophobic face of the active site, positioned within 4 of r esidues Val121, Val143, Leu198, and Trp209 (Fig ure 3 1 and 3 2, Table 3 2), and were refined, assuming full occupancy, and had final B factors of 14.0 and 15.2 2 (comparable to the protein), respectively (Table 3 1). Comparison of Holo and A po CO 2 Bound HCA II The holo HCA II structure shows, as previously modeled ( 37, 47 ) that one of the oxygens of the CO 2 O(2), interacts (3.5 ) with the amide of Thr199, and in doing so causes a displacement of the water mole cule W DW while the O(1) is positioned between
68 the zinc and Val121. This arrangement places both CO 2 oxygens nearly equidistant from the oxygen of the zinc bound solvent with distances of 3.0 and 3.1 respectively, putting the carbon 2.8 from the zinc bound solvent. This results in a side on orientation of CO 2 with the zinc bound solvent, at a distance that is well suited for the nucleophilic attack to take place on the carbon by the lone pair electrons of the oxygen in the zinc bound hydroxide (Table 3 2, Fig ure 3 1b). Additionally, a new (or displaced) water molecule, W I not previously observed in other holo HCA II structures is seen to occupy a space between Thr200 1 and the O(2) oxygen of CO 2 (Fig ure 3 1b, c and 3 4). Interestingly, the CO 2 m olecule in the apo enzyme shares a very similar geometry despi te the absence of the zinc (Figure 3 1c). A water is positioned near, what would have been the zinc bound solvent in the holo HCA II though it is ~0.6 closer to the histidine ligands. Both the CO 2 oxygens are positioned ~3.1 from this water molecule. The small shift of this water allows the CO 2 to pivot about the O(1) atom, shifting O(2) into a slightly tighter interaction with the amide nitrogen of Thr199 (3.15 for apo compared to 3.5 for holo HCA II) (Figure 3 1 b, c). Secondary CO 2 Binding Site In addition to the catalytic binding site, another CO 2 binding site (not believed to be involved in catalysis) was observed in a second hydrophobic pocket, approximately 11 away from the ac tive site (Figure 3 2) In this pocket, the CO 2 displaces the phenyl ring of Phe226, inducing a 30 tilt with respect to the plane of the ring Furthermore, this pocket lies next to Trp97, a residue that biophysical analyses have shown acts as an initiat or of proper folding of HCA II ( 128 )
69 Water Structure and a Short Hydroge n Bond The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 resolution reveals that a water molecule, termed deep water, W DW and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc bound solvent molecule, a conclusion based on the observed oxygen oxygen distance of 2.45 (Figure 3 3) This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace W DW co ntributes in significant part to the weak binding of CO 2 in the enzyme substrate complex, a weak binding that enhances k cat for the conversion of CO 2 into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pK a of the zinc bound water and to promote proton transfer in catalysis. The hydration of CO 2 to produce bicarbonate and a proton is catalyzed by the carbonic anhydrases (CAs) and plays a significant role in a number of physiological processes including respir ation, fluid secretion, and pH control. There are 14 human gene products classified as CAs including HCA II which is wide spread in tissues and heavily concentrated in red cells. The most efficient of these enzymes, together with HCA II, proceed near diffu sion control with k cat /K m for hydration at 10 8 M 1 s 1 ( 32, 92 ) Our understanding of the steps in this catalysis is based in significant part on the structure of the active site revealed by x ray crystallography studies. The first HCA II structures were determined to 2.0 resolution and identified the key features of the enzyme mechanism ( 117 ) whereas subsequent structures obtained between 2.3 and 1.1 resolution have focused on detailed understanding of the geometry about the zinc, orientations of the proton shuttle residue His64, and solvation o f residues the active site ( 34, 93, 94 ) Recent structural analysis of HCA II at 0.9 resolution reported here
70 allows enhanced interpretation with application to understanding the catalytic mechanism, in particular additional understanding of the role of solvent. A wide body of spectroscopic and kinetic data ar e consistent with a pK a near 7 describing the protolysis of the aqueous ligand of the metal forming zinc bound hydroxide ( 32, 92 ) The mechanism of catalysis comprises nucleophilic attack of zin c bound hydroxide on CO 2 followed by transfer of a proton from zinc bound water to solution to regen erate the active form A network of apparently hydrogen bonded water molecules is observed in crystal structures extending from the zinc bound solvent to t he inwardly oriented proton shuttle residue His64 located about 8 from the metal ( 93, 94 ) This structure of ordered water molecules is likely closely related to viable pathways of proton transfer during catalysis ( 129, 130 ) The crystal st ructure of human carbonic anhy drase II (HCA II) obtained at 0.9 resolution reveals that a water molecule, termed deep water, W DW and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc bound solvent molecule, a conclusion based on the observed oxygen oxygen distance of 2.45 (Figure 3 3) This water structure has simi larities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace W DW contributes in significant part to the weak binding of CO 2 in the enzyme substrate complex, a weak binding that enhances k cat for the con version of CO 2 into bicarbonate. In addi tion, this short, strong hydrogen bond is expected to contribute to the low pK a of the zinc bound water and to promote proton transfer in catalysis.
71 The final refined 0.9 resolution model, 258 residues and 486 wat er molecules, was refined to an R cryst of 12.5% and R free of 13.1 %. A full description of the structure determination and data collection and refinemen t statistics is given in Table 3 3. Of particular interest for this study is the structure of the app arently hydrogen bonded solvent water network that includes the zinc bound solvent. This network emanates from the deep water in the hydrophobic pocket formed in part by the side chains of Val121, Val143, Trp209, and Leu198 to the wate r molecules labeled W 1, W2, W3a and W3b shown in Figures 3 3. In crystal structures, this chain extends to but is not in hydrogen bond contact with the proton shuttle residue His64. The zinc bound solvent appears to form a hydrogen bond with the side chain of Thr 199, and the deep water molecule W DW appears to participate in hydrogen bonds with the backbone amide of Thr199 and with the zinc bound water molecule. The mechanism of the proton transfer utilizing pathways such as this has been the subject of considerable investigati ons ( 70, 92, 113, 129 132 ) Discussion Catalysis of the hydration of CO 2 by HCA II at 10 8 M 1 s 1 approaches the diffusion controlled limit and follows M ichaelis kinetics with a maximal turnover near 10 6 s 1 and K m near 10 mM. The diffused CO 2 is expected to be loosely bound since it has no dipole moment, and the fact that CO 2 is more soluble in organic solvents is consistent with the observed hydrophobic binding site, which suggests that solvation is a significant contributor to binding. The dissociation constant of CO 2 at the active site of HCA II was estimated by infrared spectroscopy to be 100 mM ( 48 ) a value consistent with the kinetic properties of t 2 in water under the conditions of these experiments (15 atmospheres CO 2 ) indicates a
72 maximal concentration of CO 2 near 0.45 M ( 133 ) These considerations suggest a nearly complete occupancy of CO 2 at the active site. With an energy barrier for catalysis near 10 kcal/mol, an insignificant reaction rate is expected at liquid nitrogen temperature. However, in our procedure CO 2 was intro duced to the crystal at room temperature, a procedure that surely decreased the effective pH of the crystal and surrounding solvent and promoted the formation of the zinc bound water at the active site. The observation of CO 2 at the active site is consiste nt with a zinc bound water in our structures since this form would predominate at acidic pH and is unreactive toward CO 2 The zinc bound hydroxide form of the enzyme reacts with CO 2 ; however, the observation of no bound bicarbonate suggests that this form of the enzyme was not prominent. That the binding of CO 2 does not involve first shell coordination to the zinc is consistent with previous spectroscopic studies ( 134 136 ) Moreover, the observed CO 2 binding site c onfirms previous kinetic and structural analyses of mutations made at Val143 ( 126, 137 ) (9, 29). From these studies it was shown that bulkier substitutions led to significant decreases in activity. For example a V14 3Y mutant had less than 0.02% the activity of the wild type enzyme. A structural least squares superposition of V143Y with that of CO 2 bound wild type enzyme (C RMSD = 0.26 ) clearly shows that the tyrosine would directly interfere with CO 2 binding, thu s blocking the substrate from binding in an orientation that is optimal for nucleophilic attack by the zinc bound hydroxide ( Figure 3 4 ). The binding interactions of CO 2 determined here are very similar to those of the ent inhibitor of HCA II Crystallographic analysis of
73 HCA II shows that cyanate is bound on the hydrophobic surface of the active site cavity and does not displace the zinc bound water ( 138 ) Moreover, like bound CO 2 the cyanate ion displaces the deep water and forms a hydrogen bond with the backbone amide of Thr199; the tetrahedral coordination about the zinc is not disturbed in the comple x. The distance between the carbon of bound cyanate and the oxygen of zinc bound water is 2.4 again similar to the corresponding distance for bound CO 2 This comparison of the binding of CO 2 supports our hypothesis that the observed binding site of CO 2 is a site of productive substrate binding. It is interesting to note that in studies of Co(II) substituted carbonic anhydrase, cyanate appears to bind directly to the zi nc ( 60 ) Lastly, the method of using pressurized gases, such as CO 2 may be applicable to other enzymes to capture weakly bound substrates and/or identify hydrophobic pockets in enzymes that might play important roles in substrate binding or protein folding. T he current 0 .9 resolution structure provides a clearer view of the solvati on at the active site The hydrogen bonds in this water network have distances typical of solvent water, with O O distances near 2.7 to 2.9 However, there is a short hydrogen bond with O O distance estimated at 2.45 between W DW and t he zinc bound solvent The crystallographic occupancy is near 100% for W DW and both this water molecule and the zinc bound solvent have B factors that are low (near 10 2 ) and close in value to the B factors of the surrounding amino acids. Under specific and well described conditions, short hydrogen bonds involving water with O O distances close to 2.4 have been observed ( 139 ) These are designated low barrier hydrogen bonds (LBHB) reflecting the low barrier for hydrogen
74 movement between the heteroatoms. Such LBHBs are us ually observed in nonprotic solvents and involve closely matched values of pK a for the heteroatoms of the hydrogen bond ( 139 ) The W DW is bound in a hydrophobic pocket of the active site. Moreover, with a solution pK a near 7.0, the zinc bound solvent in the crystal structure is probably in large part zinc bound hydroxide under our conditions of crystallization (pH 7.0) ( 140 ) This structure has similarities with the identification by crystallography of the LBHB of the hydrated hydroxide anion HOHOH formed in the hydrophobic region between sheets of phenyl rings in trimethyl ammonium salts of tris (thiobenzohydroximato) chromate(III) ( 141 ) In this case the heavy atom distance is 2.3 in a structure the authors describe as a central proton surrounded by two OH groups. This is p robably a good model for the observed LBHB in HCA II, in which the deep water is in a hydrophobic environment and likely involves the zinc bound hydroxide.
75 Table 3 1. Data and refinement statistics for CO 2 bound holo and apo HCA II crystal structures a values in parenthesis are for the highest resolution shell b R sym = ( | I | / ) x100; c R cryst = ( |F o | |F c | / |F obs |) x 100 d R free is calculated the same as R cryst except it uses 5% of reflection data omitted from refinement e The first number given is the average B fa ctor for the active site bound CO 2 the second is for the CO 2 bound near Phe226 f The root mean square deviation of C positions as compared to the 1.1 resolution crystal structure of unbound holo HCA II (PDB ID: 2ILI) h olo apo Space Group P 2 1 P 2 1 Cell Dimensions a, b, c () 42.4, 41.5, 72.4 42.2, 41.5, 72.3 90.0, 104.1, 90.0 90.0, 104.2, 90.0 Resolution () 20 1.1 (1.12 1.10) a 20 1.1 (1.12 1.10) b R sym (%) 8.8 (51.9) 8.0 (50.6) 21.0 (4.1) 35.6 (4.25) Completeness (%) 99.9 (100.0) 93.1 (89.7) Redundancy 11.4 (10.8) 7.0 (5.8) R efinement No. Reflections 98,494 86,919 c R cryst / d R free (%) 10.90 / 12.89 10.35 / 13.87 No. Atoms Protein 2096 2121 Zinc/CO 2 /glycerol 1/6/12 0/6/6 Water 404 352 B factors ( 2 ) Protein (main / side) 10.4 / 15.2 11.1 / 15.6 Zinc/CO 2 e /glycerol 5.1/14.0, 39.5/20.1 NA/15.7, 59.7/19.1 Water 31.7 29.4 Ramachandran Plot (%) Allowed 89.4 88.9 Additionally allowed 10.2 10.6 Generously allowed 0.5 0.5 f RMSD 0.192 0.144
76 Table 3 2 Distance () geometry of CO 2 for holo and apo HCA II holo apo Zn bound OH / H 2 O C 2.8 H 2 O C 2.9 Zn bound OH / H 2 O O(1) 3.0 H 2 O O(1) 3.1 Zn bound OH / H 2 O O(2) 3.1 H 2 O O(2) 3.1 Zn +2 O(1) 3.2 Thr199(N) O(2) 3.5 3.2 W I O(2) 3.5 3.2 His119 O(1) 3.4 3.5 O(2) 3.4 3.5 O(1) 3.3 3.2 W I C 3.6 3.2 O(1) 3.5 3.6 O(1) 3.5 3.4 O(2) 3.5 3.3 O(1) 3.6 3.5 O(1) 3.7 3.8 O(2) 3.7 4. 1 Zn +2 C 3.7 O(1) 3.7 3.8 C 3.9 3.9 C 3.9 3.9 The bond distances from the CO 2 molecule are given within a radial shell of 3.9 The numbering of the CO 2 oxygens are in accordance to the text and figures. The water molecule in the apo hCA II is in an equivalent position to that of zinc bound OH / H 2 O in the holo hCA II.
77 Table 3 3 Refinement and model statistics for 0.9 HCA II crystal structure Data collection statistics Space Group P2 1 Unit cell p arameters (,) a = 42.2 b = 41.3, c = 72.2 =104. 2 Resolution () 50 0.90 (0.92 0.90)* R sym 0.078 (0.58) Completeness (%) Redundancy 25.0 (2.0) 92.3(76.1) 6.1 (2.8) Total number of unique reflections 164840 (6751) a R cryst (%) b R free (%) 12.5 13.1 Residue Nos. 4 261 No. of prote in atoms 2327 No. of H 2 O molecules 487 B factors ( 2 ) Aver age, main chain, side chain, Zn, solvent 10.9 15.26 5 .0, 26.0 Ramachandran statistics (%) Most favored, additionally allowed and generously allowed regions 88, 11.5, 0.5 R.m.s.d. for bond le ngths (), angles () 0.03, 1.0 Values in parentheses refer to the highest resolution bin. a R symm b R cryst F o| | F F obs | x 100 c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement.
78 Figure 3 1. HCA II structure. (a) Overall view, showing the hydr ophilic (magenta stick representation) and hydrophobic (green surface representation) sides of the active site. The active site zinc is shown in purp le with the waters of the proton wire shown as small, red spheres. A close up stereoview of the active si te showing the position of bound CO 2 in (b) h olo and (c) apo HCA II Electron density of the active site amino acids and W I (sigma weighted 2F o F c 2 (sigma weighted F o F c Fourier map (www.pymol.org).
79 Figure 3 2. Second CO 2 binding site. (a) Surface representation showing the separation of the active site (green) and non catalytic (pink) CO 2 binding pockets. (b) Close up view of the CO 2 binding. Note the conformational change in Phe226 (red = unbound, green = CO 2 bound holo h CAII). The electron density is a sigma weighted 2F o F c Fig u re created using PyMOL (www.pymol.org).
80 Figure 3 3. The ordered water network in the active site of HCA II. The zinc is represented by a gray sphere and the oxygen atoms of water molecules as smaller red spheres. Dotted lines are presumed hydrogen b onds with heavy atom distances given. Stick figures are selected amino acids of the active site with both the inward and outward orientations of His64 shown. This figure was created using PyMOL.
81 CHAPTER 4 ROLE OF HYDROPHILIC RESIDUES IN THE EXTENDED ACTIV E SITE Introduction The rate limiting step in maximal velocity of catalysis in HCA II is the transfer of a proton between His64 and the zinc bound solvent ( 42, 142 ) His64 is located on th e side of the active site cavity with its side chain extending into the cavity (Figure 4 1) ( 34, 94, 117 ) In crystal structures, two orientations are observed for this side chain, both of which are about equa lly populated at pH near 7 ( 93, 94 ) One is an inward conformation with the side chain oriented towards the zinc, and a second is an o utward conformation with His64 oriented towards the mouth of the active site cavity and external solution. is too far from the zinc (about 7.5 ; ( 51, 93 ) ) for direct proton transfer, and solvent hydrogen isotope effects are consistent with proton transfer through intervening hydrogen bonded solvent bridges ( 143 ) Crystal structures of the isozymes of CA in the rogen bonded water molecules between His64 and the zinc bound water in t he active site cavity In the Grotthuss mechanism, high proton mobility is achieved by proton shuttling along hydrogen bonds without requiring much motion from their oxygen atoms. Howe ver, the apparently hydrogen bonded water structure shows at best weak hydrogen bonding between the nearest water molecule W2, a di The proton migrati on in CA is of great current interest as a model for proton transfer in more complex systems, and is also under study in several labs using computational methods ( 76, 130, 131, 144, 145 )
82 These co ntinuing studies of CA are significant in understanding the role of water in facilitating proton transfers in proteins and in catalysis. Recent attention has focused on the significance of the ordered solvent observed in the crystal structures of CA. Sev eral residues have been shown to stabilize this ordered solvent structure, among them Tyr7, Asn62, and Asn67 (Figure 4 1) ( 113, 146 ) Fisher et al. ( 113 ) replaced these hydrophilic residues with hydrophobic residues (Y7F, N62L, and N67L) and observed several changes in structure and catalysis. These mutations had different effects on the orientation of the side chai n of His64 (Y7F and N62L predominantly inward and N67L predominantly outward), altered the pK a of His64, and affected the ordered water structure. The observed rate constants for proton transfer between His64 and the zinc bound hydroxide were changed for t hese mutants, but there was little effect on the rate constant for conversion of CO 2 into bicarbonate ( 113 ) Among these the mutant N62L was interest ing since the X ray crystal structure showed His64 in the inward orientation and the ordered water structure intact as in the wild type ( 113 ) The s ide chain of Asn62 in HCA II is extended into the active site cavity about 9 from the zinc and as close at 3.3 from the side chain of His64 ( 93, 94, 117 ) This residue appears conserved in many species of CA II, for example in mouse and chicken as well as in many other is ozymes of carbonic anhydrase ( 147 ) Computations of conformational states of active site residues in HCA II point out ste ric interactions between the side chains of His64 and Asn62 that contribute to the orientation of His64 ( 130, 146 ) In previous experimental studies, the pH profile of ca talysis by N62L HCA II appeared irregular and difficult to interpret ( 113 ) However, additional amino acid
83 substitutions at position 62 in this study have provided a more straightforward interpretation. The replacement of Asn62 with other amino acids was shown to cause changes in the orientation of the proton shuttle His64 and the pK a of its imidazole side chain, but had little or no effect on the stru cture of ordered solvent in the active site cavity. The results point out the capacity of His64 to participate in proton transfer more efficiently in the inward than in the outward conformations, and the role of residue 62 in fine tuning the values of pK a of His64 and the zinc bound solvent molecule. In addition to Asn62, Tyr7 also lies adjacent to His64 in the hydrophilic side of the active site cavity. It is conserved in the mammalian CAs as well as found in other lass ( 147 ) The side chain of Tyr7 in HCA II extends into the active site cavity with no apparent interactions with other residues (Figure 4 1). The hydroxyl of Tyr7 appears to be within hydrogen bonding distance of water molecule W3a; however, the neutron diffraction structure at a crystallization pH of 9 shows no such hydrogen bond ( 66 ) Here we discuss a number of substitutions at position 7 and 62 in HCA II to provide further insight to the structural aspects influencing the rate of the intramolecular proton transfer step in the catalysis. Initial studies by stopped flow spectropho tometry using the mutant Y7F HCA II showed catalytic activity in hydration marginally reduced ( 148 ) However, further examination using 18 O exchange showed that the proton transfer component of catalytic dehydration was enhanced as much as 7 fold compared to wild type ( 113 ) Catalysis by each of the variants was studied by esterase activity and by 18 O exchange betwee n CO 2 and water using membrane inlet mass spectrometry. The x ray crystal structures of N62A, N62D, N62V, N62T and Y7I HCAII were determined at 1.5
84 1.6 resolution. We have found that substitution at Tyr7 had no effect on the first stage of catalysis (Eq. 4 1), but certain replacements at position 7 showed rate constant for proton transfer enhanced nearly 10 fold compared with wild type. The variant Y7I HCA II had a lower thermal stability and an altered conformation in the first eleven residues at the ami no terminus. These studies emphasize the role of Tyr7 in establishing the fold of the N terminus of HCA II and its influence in long range, intramolecular proton transfer. Methods and Materials Express ion and Purification of HCA II M utants Asn62 was repl aced with Asp, Ala, Val and Thr. Tyr7 was replaced with Ala, Ile, Trp, Asp, Asn, Ser, and Arg ( 42 ) Rose Mikulski of the Silverman lab mutated, expressed and purified the variants at position 7. All t he point mutations of this study were generated by site directed mutagenesis using the QuikChange II Kit (Stratagene, LaJolla). DNA sequ encing over the entire coding region of HCA II confirmed the single mutants. Expression of each mutant was done by transforming mutated vectors into Escherichia coli BL21(DE3)pLysS cells, which do not express any indigenous CA under the following condition s. The transformed cells were expressed at 37 C in LB medium containing 100 g/ml ampicillin. HCA II production was induced by the addition of isopropyl thiogalactoside to a final concentration of 1mM when the bacterial culture reached an OD 600 of 0.6. Th e cells were harvested 4 hrs after induction. The cell pellets were lysed and HCA II was purified through affinity chromatography using p (aminomethyl)benzenesulfonamide coupled to agarose beads ( 149 ) Electrophoresis on a 10% polyacrylamide gel stained with Coomassie Blue was used to confirm the purity of enzyme samples, which were found to be greater than 96% pure. HCA II and the mutants studied here bound sulfonamides tightly; therefore, enzyme concentrations
85 were determined by titration of active sites with ethoxzolamide while measuring the ca talyzed depletion of 18 O from CO 2 and analyzing data with the Henderson approach ( 150 ) Crystall ization of N62 Mutants Crystals of the HCA II single site mutants of N62A, N62V, N62T and N62D were obtained using the hanging drop vapor diffusion method ( 151 ) The crystallization drops HCl (pH 7.0) with 5 HCl (pH 9.0) and 1.3 M sodium citrate at 20 C against 1 ml of the precipitant solution. The pH of the crystallization solutions was about 7.9. Useful crystals were observed 4 days after the crystallization setup. Previous studies had shown the binding of sulfate at the zinc in N62L HCA II ( 113 ) ; hence sulfate was avoided in crystallizations reported here. Crystall ization of Y7I HCA II After repeated attempts to crystallize mutants Y7A, Y7W, Y7D, Y7N, Y7R and Y7S, we were successful only with Y7I and Y7F HCA II; the crystal structure of the latter was reported earlier ( 113 ) One may speculate that mutations at Try 7 produce a disordered N terminus giving rise to structural heterogeneity in the sample, which is likely to deter the crystal nucleation or crystal growth. Crystals of the HCA II Y7I mutant were obtained using the hanging drop method ( 151 ) ~10 mg/mL in 10 mM Tris sodium citrate 100 mM Tris Cl (pH 8.0)) against a well of 1 mL precipitant solution. A few crystals were observed about a month after the crystallization setup at 293 K.
86 X ray Diffraction and Refinement The crystals of all the above mutant forms of HCA II were isomorphous with the wild type enzyme with mean unit cell dimensions of a = 42.7 0 .1 b = 41.7 0.1 c = 72.8 0.1 and = 104.6 0.1 (Table 4 1, Figures 4 2, 4 3). All data sets were greater than 92% complete and were processed to 1.7 1.5 resolution (Table 4 1). A least squares superposition of these mutants with the crystal structure of wild type HCA II (PDB ID: 2C BA; ( 51 ) ) gave an average RMSD of 0.09 0.0 4 T he polypeptide backbone at position 62 was shifted slightly into the active site cavity for the mutants replacement of Asn62 with Asp and Ala induced shifts in the side chains of nea rby residues Asn67 and Gln92 1 2 of N62A, the same atoms were shifted by 0.8 and 0.2 respectively towar ds His64 1 2 3 3 The X ray diffraction dataset for all of the mutant HCA II crystals were obtained at room temperature, using an R AXIS IV ++ image plate system with Osmic mirrors and a Rigaku RU H3R Cu rotating anode operating at 50 kV and 100 mA. The detector crystal distance was set to 80 mm. Each dataset was collected at room temperature with the crystals mounted in qua rtz capillaries. The oscillation steps were 1 with a 7 min exposure per image. X ray data processing was performed using DENZO and scaled and reduced with SCALEPACK ( 152 ) All manual model building was performed using Coot ( 112 ) and refinement was carried out wi th the crystallography and nuclear magnetic resonance system (CNS) suite of programs, version 1.1 ( 153 )
87 The wild t ype HCA II crystal space group ( PDB ID: 2CBA ( 51 ) ) was isomorphous with all of the dataset collected, and was used to phase the dataset To avoid phase bias of the model, the zinc ion, mutated residues, and water molecules were r emoved. After one cycle of rigid body refinement, annealing by heating to 3000 K with gradual cooling, geometry restrained position refinement, and temperature factor refinement, 2 F o F c and F o F c Fourier electron density maps were generated. These elec tron density maps clearly showed the position of the zinc and mutated residues, which were subsequently built into their respective models. After several cycles of refinement, solvent molecules were incorporated into the models using the automatic waterpic king program in CNS until no more water molecules were found at a 2.0 level. Refinement of the models continued until convergence of R cryst and R free was reached ( Table 4 1). Oxygen 18 Exchange The 18 O experiments for the HCA II variants of this study were conducted in the Silverman lab; Rose Mikulski performed those for Tyr7; Jaiyin Zheng and Chingkuang Tu performed those for Asn62. This method is based on the measurement by membrane inlet mass spectrometry of the depletion of 18 O from species of CO 2 ( 55, 154 ) A continuous measure of isotopic content of CO 2 is provided by CO 2 passing across the membrane wher e it enters a mass spectrometer (Extrel EXM 200). In the first of two independent stages of catalysis, the dehydration of labeled bicarbonate has a probability of transiently labeling the active site with 18 O (E q 4 1 ). In a second stage, the protonation o f the zinc bound 18 O labeled hydroxide results in the release of H 2 18 O to the solvent (E q 4 2 ) HCOO 18 O + EZnH 2 O EZnHCOO 18 O COO + EZn 18 OH ( 4 1 ) H + His64 EZn 18 OH His64 EZnH 2 18 O His64 EZnH 2 O + H 2 18 O (4 2 ) H 2 O H 2 O
88 Two rates for the 18 O exchange catalyzed by carbonic anhydrase are obtained by this method ( 55 ) The first is R 1 the rate of exchange of CO 2 and HCO 3 at che mical equilibrium, as shown in E q 4 3 Here k cat ex is a rate constant for maximal interconversion of substrate and product, K eff S is an apparent binding constant for substrate to enzyme, and [S] is the concentration of substrate, either CO 2 or bicarbonate ( 155 ) The ratio k cat ex / K eff CO2 is, in theory and in practice, equal to k cat / K m for hydration obtained by steady state methods. R 1 /[E] = k cat ex [CO 2 ]/( K eff CO2 + [ CO 2 ]) (4 3) R H2O, the rate of release from the enzyme of w ater bearing s ubstrate oxygen (E q 4 4 ), is the second rate determined by the 18 O exchange method. This is the component of the 18 O exchange that is dependent upon the donation of protons to the 18 O labeled zinc bound hydroxide ( 42, 55 ) In such a step, His64 as a predominant proton donor in th e catalysis provides a proton (E q 4 2). In E q 4 4 k B is the rate constant for proton transfer to the zinc bound hydroxide and ( K a ) donor and ( K a ) ZnH2O are the ionization constants of the proton donor and zinc bound water molecule. The determination of the kinetic constant k B and ionization constants of E q 4 4 was carried out by nonlinear least squares methods (Enzifitter, Elsevier Biosoft, Cambrige, U.K .) k B obs = k B /([1+( K a ) donor /[H + ] [1+[H + ]/( K a ) ZnH2O ]) ( 4 4 ) The catalyzed and uncatalyzed exchange of 18 O between CO 2 and water at chemical equilibrium were measured in the absence of buffer at a total substrate concentr ation of 25 mM using membrane inlet mass spectrometry ( 55 ) T he temperature was 25 C and the total ionic strength of solution was kept at a minimum of 0.2 M by the addition of Na 2 SO 4
89 Esterase Activity These esterase experiments were conducted in the Silverman lab; Rose Mikulski performed those for Tyr7 mutants; J aiyin Zheng and Chingkuang Tu performed those for Asn62 mutants. The hydrolysis of 4 nitrophenylacetate uncatalyzed and catalyzed by variants of carbonic anhydrase was measured by the method of Verpoorte et al. ( 156 ) The increase in absorbance at 348 nm was measured; this is the isosbestic point of the product nitrophenol and the nitrophenolate anion with an absorbtivity of 5.0 x 10 3 M 1 cm 1 Initial velocities were me asured using a Beckman Coulter DU 800 spectrophotometer. Differential Scanning Calorimetry (DSC). DSC experiments were performed using a VP DSC calorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of ~0.5 ml. V ariants of HCA II were buffered in 50mM Tris H m ) of unfolding were calculated by integrating the area under the peaks in the thermog rams after adjusting the pre and post transition baselines. The thermograms were fit to a two H vH ) of unfolding ( 157 ) Role of the Water Structure and Orientation of His 64 We examined the crystal structures a nd catalytic properties of mutant s of HCA II containing substitutions at position 7 and 62, namely, Y7I N62A, N62D, N62V, and N62T. The side chain of the proton shuttle His64 is of particular interest and its dual orientation in HCA II is well documented ( 93, 94 ) The inward and outward conformers of His64 in the wild type structure of Fisher et al (82) were refined to occupancy of
90 approximately 70% and 20% respectively, whereas these conformers of His64 in N62T were refined with equal occupancy of 50% each. Ful l occupancy was assigned to His64 in the case of the mutants that report only a single conformer of His64 (N62A,V,L), alth alternative conformation. Appropriate B factors are given in Table 4 2. 1 and 2 near 44 and 95, respectively, while the outward orientation has angles of 39 and 98. The 1 describes the inward and outward orientations of the side chain of was observed only in the in ward conformation, and for N62D only in the outward conformation In contrast, the mutant N62T HCA II resembled wild type enzyme by displaying two superimposed conformations for the His64 side chain, the inward and outward. These two side chain conformatio ns were equally present in the crystal structure of N62T HCA II (Table 4 2, Figure 4 2) None of the mutations significantly disrupted the active site solvent network compared with wild type HCA II, although a slight shift of 0.7 in the position of the water molecule W3b was observed in all the mutant forms (Figures 4 2, 4 3). In the case of the hydrophobic substitutions of Ala and Val at position 62, water molecule W3b moved away from the side chain of residue 62 (Figures 4 3A and B). Hydrophilic substi tutions of Asp and Thr caused W3b to move towards the polar side chain of residue 62 (Figures 4 3C and D). Besides these small positional perturbations, the network of ordered solvent remained conserved (Figure 4 3). Of all the mutant
91 structures, N62A has slightly higher B factors for the ordered water structure, but this is most likely due to its slightly l ower resolution than the others. The structures of Y7I and Y7F HCA II are similar to wild type in that the residues coordinating the zinc and those in the active site cavity (with the exception of residue seven) are unchanged (Figures 4 1, 4 4). Moreover, both show His64 predominantly in the inward orientation (Figure 4 4). However, the structure of Y7I HCAII showed several interesting features not prese nt in wild type or Y7F HCA II. Most notably there was a novel fold in residues 4 to 11 of the N terminal chain (Figure 4 5); while the amino terminal residues 1 4 residues were not observed in the crystal structure presumably because of disorder. This N te rminal fold in Y7I HCA II caused the side chain of Ile7 to point away from the metal in the active site; the side chain of Ile7 does not occupy a position analogous to the side chain of Tyr7 in wild type, which lies between Thr200 and His64 (Figure 4 4A, 4 5). Despite the differences in structure of the N terminal residues of Y7I and wild type, the geometry of the residues coordinating the zinc and the extended water structures of the active site are not altered (Figures 4 4A). Asn62 and Tyr7 Contribute to Facile Proton Transfer The catalysis by carbonic anhydrase of 18 O exchange between CO 2 and water yields two rates, as described in Methods. The first is R 1 of E q 4 3 from which we determined k cat ex /K eff CO2 for the hydration direction. The pH profile of t his rate constant for wild type and mutants at residue 62 were adequately fit to the titration of a single residue (Figure 4 6 ) with resulting values of the kinetic pK a an estimate of the pK a of the zinc bound water ( 32, 155 ) given in Table 4 3. These data show a maximum at high pH correspo nding to the reactivity of the zinc bound hydroxide form of the enzyme in the hydration direction, with the maximal values of k cat ex /K eff CO2 given in Table 4 4. The pK a
92 values were all close to pK a 7.0 as found in wild type, with the exception of N62D HCA II for which the pK a was elevated to 7.9 (Table 4 3, column 5). However, the maximal, pH independent values of k cat ex /K eff CO2 for the four mutants showed considerable variation with each mutant less than k cat ex /K eff CO2 for wild type; the value for N62D was the smallest (Table 4 4 ). A somewhat more precise fit of the data was achieved by introduction of a second ionization; however, this had minor effects on the resulting parameters of Tables 4 3 and 4 4. The values of K eff CO2 are large in these experiments, exceeding the solubility of CO 2 (about 34 mM) under our conditions. Hence, k cat ex was not able to be determined. The values of pK a determined from k cat ex /K eff CO2 in the 18 O exchange experiment are very similar to the values of pK a determined from the pH d ependence of k cat /K m for the catalyzed hydrolysis of 4 nitrophenylacetate (Table 4 3, column 5). The variation in the maximal values of k cat /K m for ester hydrolysis was less that for k cat ex /K eff CO2 ; however k cat /K m for N62D HCA II was the smallest in bo th sets of data (Table 4 4). Again, K m for this ester hydrolysis is too large to determine k cat The values of R H2O /[E] provide an independent set of parameters that represent a rate constant for the proton transfer dependent release of H 2 18 O from the acti ve site ( E q 4 4). R H2O /[E], the rate of release of H 2 18 O from the active site, is determined by intramolecular proton transfer as verified by pH profiles, kinetic isotope effects, and chemical rescue experiments ( 154, 158 ) The pH profiles of R H2O /[E] appear bell shaped for much of the pH range of these studies (Figure 4 7 ), a feature that ha s been interpreted in terms of E q 4 4 and the transfer of a proton from one prominent donor His64 to the zinc bound hydro xide ( 154, 158 ) The solid lines for the bell shaped
93 regions of Figure 4 7 represent fits of E q 4 4 to the data with the resulting parameters appearing in Tables 4 3 and 4 4. In order to apply E q 4 4 correctly, the assignment of the values of pK a for the donor and acceptor needs to be established. This was achieved using the values of pK a for the zinc bound water pK a ZnH2O as determined by k cat ex /K eff CO2 from the 18 O exchange experiment and by k cat /K m for the es ter hydrolysis, which are in agreement (Table 4 3, columns 5,6). These experiments were carried out at chemical equilibrium in the absence of added buffer. Buffers can participate in the proton transfer and complicate interpretation. The values of pK a ZnH2 O and pK a His64 for wild type and N62T HCA II were identical (Table 4 3); however, the value of the rate constant for proton transfer k B for N62T is half that of wild type (Table 4 4). For N62D HCA II the value of pK a ZnH2O is elevated to 7.6 7.9 (Table 4 3) with the value of pK a His64 at 8.4 also elevated compared with wild type. This corresponds to His64 predominantly in the outward orientation with k B very 1 about 5% of the value of wild type (Table 4 4). The remaining variants of Tables 4 3 and 4 4, specifically N62A, V and L, are not as straightforward because the values of pK a ZnH2O near 7.2 determined from k cat ex /K eff CO2 of the 18 O exchange experiment and k cat /K m from the ester hydrolysis do not agree with the values of pK aZnH2O determined from R H2O /[E] that are near 6.0. The bell shaped curves for these variants are clearly shifted to lower pH (Figure 4 7 ) consistent with values of pK a ZnH2O and pK a His64 both near 6.0 (Table 4 3). The resulting values of k B ar 1 not very different than the corresponding values of the other enzymes of Table 4 4 (except N62D). The data for N62L HCA II are from a previous report ( 113 ) and were not interpreted there because of their complicated pH
94 profile for R H2O /[E]. However, the data of Figure 4 7 show pH profiles for N62A and N62V that are less complicated but show features shared by N62L. Wi th this background E q 4 4 was applied to a ll three of these variants N62A, V, and L. We note that each of the mutants N62A and N62V (Figure 4 7 ), as well as N62L ( 113 ) have a secondary, very small maximum for R H2O /[E] at pH near 8 that resembles the data for N62D. This presumably indicates a second, less efficient proton transfer pathway. The pH profiles for k cat exch /K eff CO2 by the Tyr7 mutants under study were also adequately fit to a single ionization and appeared similar to that of the wild type (Figure 4 8 ). The resulting maximal values of k cat exch /K eff CO2 represent catalytic activity in the hydration direction and were essentially identic al to th at of wild type (Table 4 5 ). The variation was greater for the activities k cat /K m for the catalyzed hydrolysis of p nitrophenylacetate (Table 4 5 ). This probably reflects the larger size of the substrate for the ester hydrolysis which is influenced more by changes at residue 7. T he resulting values of pK aZnH2O representing the ionization of the zinc bound water for each mu tant have been listed in Table 4 6 The data demonstrate that the values of pK a ZnH2O of the zinc bound water are near 7 for wild type an d mutants. These pK a ZnH2O values were confirmed by measurement of esterase activity (Table 4 6 ). The pH profiles of the rate constant R H2O /[E] for variants at position 7 (Figures 4 9 ) provide three constants relevant to the c atalysis, according to Eq 4 4 The first are estimates of pK a ZnH2O which are given in Table 4 6 and generally are consistent with the values described in the above paragraph. Exception s are Y7A, Y7S, and Y7R (Table 4 6 ). These differences may be due to different properties of the act i ve site for the processes of E q 4 1 and E q 4 2 or possibly the irregular curves for R H2O during
95 catalysi s by Y7A and Y7S In later construction of a free energy plot, we have used data only from R H2O The second constants are the values of pK a His64 (Ta ble 4 6 ). These values are uniformly lower for replacements at residue 7 than the value of pK a His64 at 6.9 in wild type HCA II. Such a shift in pK a His64 is associated with the inward coordi nation of the side chain of His 64 and its more hydrophobic enviro nment than in wild type HCA II in which this side chain appears about equally in inward and outward orientations ( 93, 94 ) The third c onstant is the maximal, pH independent value of k B describing intramolecular proton transfer in the dehydration direction ob tained by a fit to eq 6 (Table 4 5 ). The pH profiles for R H2O from which k B values were obtained, were more difficult to fit for Y7 D and Y7N than the bell curve of wild type HCA II and other variants. The data for Y7I were consistent with a value of k B 1 from a fit of Figure 3. This value is greater 1 for wild type, although their curves for RH2O appear s imilar, because of the larger difference in pKa between donor and acceptor for Y7I. The greatest change in k B 1 (Table 4 5 ). The thermal stability of Tyr7 mutants was determined by DSC A single p eak representing the main unfolding transition was observed for all of the variants of Table 1 and is shown for wild type, Y7I H CA II and Y7F HCA II in Figure 4 10 The fit to these data was based on a two state, reversible unfolding model to obtain the th ermodynamic parameters. The main unfolding transition of the wild type enzyme T m occurred at 59.5 0.5 C and that of Y7I occurred at 51.8 0.5 C. The values of T m for each of the remaining variants of Table 1 were between 49.3 and 52.5 C except Y7F wh ich had a
96 distinctly broad transition with a T m of 55.8 0.5 C. The calorimeteric parameters determined from the DSC experiments and the calculated thermodynamic parameters for all the enzymes examined are listed in Table 4 7 Discussion The goal of thi s work was to elucidate the role in catalysis of residue 62 and 7 on the hydrophilic side of the active site cavity in HCA II, in particular its influence on the structure of ordered solvent, on the properties of the proton shuttle His64, and on the kineti cs of catalysis. An advantage with the amino acid substitutions we made at position 62 and 7 was that each of the resulting mutants had an ordered solvent structure in the active site cavity very similar to that of wild type HCA II. This appears to remove solvent structure as a variable in comparing catalysis by these mutants. As a result, these mutants allow this study to focus more completely on the properties of the side chain of His64. The crystal structures of the variants at residue 62 are closely sup erimposable with wild type HCA II (Figure 4 3). A major difference is in the orientation of the side chain of the proton shuttle residue His64 which appears for certain mutants in an entirely inward or entirely outward conformation, within experimental err or (Figure 4 2 ). In wild 1 of about 100. In any case, the kinetic barrier between inward and outward orientations is near 6 kcal/mol in wild type HCA II according to computations, and is not expected to co ntribute to rate of catalysis (with an energy barrier near 10 kcal/mol) ( 74 ) For the mutation N62D HCA II, His64 was observed entirely in the outward conformation, within experimental uncertainty (Figures 4 2, 4 3). In this case the pK a for the imidazole of the side chain of His64, determined from pH profiles of catalysis, was
97 elevated to 8.4 compared with wild type which is near 7.2 (Table 4 3) ( 159 ) The elevated pK a of His64 in the N62D mutants is likely due to the nearby Asp62, possibly in an undetected inward orientation. This i s also consistent with the pH dependence of rotamer populations in wild type HCA II showing the outward orientation favored at lower pH ( 93, 94 ) In N62D HCA II, the pK a of the zinc bound water is also elevated to 7.6 7.9 (Table 4 3), which is also anticipated based on the shift to the outward orientation of the partially positively charged side chain of His64. The ordered water struc ture appears unchanged in N62D compared with wild type although His64 is in the outward orientation in this mutant (Figure 4 2). It is significant pK a between proton donor and acceptor (pK a ZnH2O a His64 ) is small, near 0.4 0.8, for both wild type and N62D HCA II, yet the rate constant for proton transfer k B for N62D is about 20 fold less than wild type (Table 4 4). This shows that the lower rate constant k B for N62D is not due primarily to changes in pK a but rather to the much lower capacity for proton transfer of His64, presumably because of its greater distance from the zinc (about 12 ). This is consistent with accum ulated evidence from other studies of the distance dependence of the proton transfer in carbonic anhydrase ( 92 ) The longer distance between His64 and the zinc bound hyd roxide in N62D (~ 12 ) is suggested to have a slower proton transfer at least in part because of the energy inherent in constructing longer water wires for proton transfer ( 74 ) In addition, computations show that the outward orientation of His64 is associated with short er lifetimes of the ordered water molecules in the active site cavity ( 146 ) Hence, the lower rate constant for proton transfer k B for N62D can be attributed to His64 in the outward orientation. However, data on this topic are complex. Some computations indicate that
98 proton transfer in c arbonic anhydrase is dominated by electrostatics and is not expected to be dependent on distance ( 70, 145 ) Introducing a charged group such as Asp62 near the proton transfer pathway would be expected to alter the electrostatic properties of the donor and acceptor, as noted above, but also the apparent pK a values of water molecules in the proton transfer pathway. This could contribute to the low efficiency of proton transfer in catalysis by N62D. The orientation of His64 in T200S HCA II is outward yet the steady state constants for catalysis are very similar to those of wild type HCA II ( 160 ) For each of the three mutants in which His64 is in the inward orientation (N62A, N62V, N62L) the pK a for His64 determined from R H2O is decrease d to near 6.0 (Table 4 3), due in part to the position of the imidazole ring in a more hydrophobic environment and more sequestered from bulk solvent (Figure 4 2). The pK a for the zinc bound water determined in these mutants from R H2O /[E] is considerably s maller than in wild type (Table 4 3). This decrease is expected in these three mutants because of greater stabilization of the zinc bound hydroxide by the inward oriented imidazolium form of His64. However, in contrast to the smaller value of pK a near 6 de termined from R H2O /[E], the pK a values for zinc bound water observed from k cat ex /K eff CO2 and of k cat /K m of the esterase catalysis are near 7 (Table 4 3). These data imply that in the transition state for proton transfer (in N62A, N62V, N62L), the pK a valu es of the proton acceptor is low compared with the pK a determined from the first stage of catalysis (E q 4 1 ). In each case however, the rate constant for proton transfer appears close to the same (0.20 to 1 Table 4 4). This probably indicates tha t it is the inward orientation that is
99 responsible for proton transfer between His64 and the zinc bound solvent molecule in catalysis. Finally, the values of pK a for donor and acceptor for N62T HCA II appear very close to those for wild type (Table 4 3). Both of these enzymes show appreciable occupancy of His64 in the inward and outward orientations. Perhaps it is significant that these pK a values are midway between those found for His64 in the mutants for which this residue lies entirely in the inward and entirely in the outward orientations. However, the rate constant for proton transfer k B is less by about two fold for N62T compared with wild type (Table 4 4). An interesting feature of the data of Figure 4 6 compared with an earlier study ( 113 ) is that in this study replacement of Asn62 with hydrophobic residues caused substantial changes in k cat ex /K eff CO2 (Table 4 4). In the case of N62D, a decrea se as much as two fold in the maximal values of k cat ex /K eff CO2 for hydration was observed (Table 4 4). This is not explained for N62A,V, and L by values of the pK a of the zinc bound water which are arguably the same as in wild type. Moreover, the decrease in k cat ex /K eff CO2 for N62D is associated with a more basic pK a for the zinc bound water than in wild type (Table 4 3). Since these mutations alter the charge density in the active site cavity, electrostatic effects on the transition state for the nucleophi lic attack on CO 2 or on the dissociation of bicarbonate could be responsible. Replacement of Asn62 with a number of amino acids maintained the order ed water structure observed by X ray crystallography in the active site cavity of HCA II. This removed water structure as a variable in comparing with wild type the activities of mutants at residue 62. A significant role of Asn62 in HCA II is to permit two
100 conformations of the side chain of His64, the inward and outward, that are necessary for maximal efficiency of this enzyme in transferring protons between the active site and solution ( 92, 161 ) The site specific mutant N62D had an entirely outward orientation of His64, yet the difference in pK a between the proton donor His64 and zinc bound hydroxide was near zero, as in wild type HCA II. The rate of proton transfer in catalysis by N62D HCA II in the dehy dration direction was 5% that of wild type, showing that His64 in this mutant is inefficient in proton transfer compared with wild type because of its predominantly outward orientation compared with wild type, which shows both inward and outward orientatio ns. Additional contributions to inefficient proton transfer could arise from nonspecific electrostatic effects due to introduction of a charged group Asp62 near the proton transfer pathway. These results emphasize the role of Asn62 among the residues on th e hydrophilic side of the active site cavity in maintaining efficient catalysis by CA In the case of mutations at position 7, the rate constants k cat exch /K eff CO2 for the first stage of catalysis (Eqs 4 1, 4 3; Table 4 5) remain unchanged. This appears to be a result of the substitutions being at least 7 away from the zinc where the interconversion between CO 2 and bicarbonate occurs. However, among the variants of Table 4 5, there is nearly a 10 fold variation in the rate constant k B for proton transfer f rom His64 to the zinc bound hydroxide in the dehydration direction. This supports a role for Tyr7 not only in fine tuning the rate of proton transfer but also in proper folding as supported by the following observation. The single replacement of Tyr7 with Ile caused a major alteration in the confirmation of the N terminal chain in the Y7I structure (Figure 4 5). The amino terminal 11 residues were displaced with respect to their
101 conformation in wild type with the side chain of Ile7 pointing away from the ac tive site, different than for Tyr7 in wild type. Yet the rate constant for proton transfer for Y7I HCA II was enhanced nearly 3 fold compared with wild type. Despite the large changes in the N terminal region of Y7I HCA II, the structure of the active sit e is remarkably similar to the structure of the wild type enzyme including the network of ordered water molecules (Figures 4 1, 4 4), unlike Y7F HCAII in which water molecule W3a is not observed ( 113 ) It is possible that the altered orientation of the N terminus of Y7I HCA II is a consequence of crystal contacts, although this mutant crystallizes in the same space group as the wild type enzyme. Trunca tion of as many as 24 residues of the N terminal end of HCA II does not prevent the remaining protein from folding correctly ( 162 ) The structure of the N terminus has been shown to form very late in folding ( 162 ) A comparison of the characteristics of Y7I and Y7F HCA II reveals that they both a ZnH2O pK a His64 ) near 1.0, and both have nearly identical values of k cat exch /K eff CO2 However, they differ considerably in their values of k B the rate constant for proton transfer in the dehydration direction, as shown in Table 4 5. This difference is shown in a free energy plot based on k B in Figure 4 11. The open circles are rate constants for proton transfer during catalysis by H64A HCA II determined by enhan cement of catalysis when proton donors are exogenous derivatives of imidazole and pyridine ( 158 ) These data are fit by Marcus theory applied t o proton transfer ( 75, 163 ) represented by the solid line of Figure 4 11 We assume this line represents the dependence of k B a within the active site. The significance of this fit is that these values of k B are for proton transfer from donors
102 not attached to the enzyme through chemical bonds, free of many restraints. Interestingly, the values of k B for wild type HCA II and nearly all of the variants of Table 4 5, except Y7F HCA II, fall on or near this Marcus line (Figure 4 11). We suggest that the reason the value of k B for Y7F lies considerably above the Marcus line of Figure 4 11 while Y7I and other mutants lie c loser to the line is the abbreviated water structure of Y7F. A significant difference is that Y7F has an unbranched, hydrogen bonded water structure in the active site cavity (Figure s 4 1, 4 4 ) compared to the wild type and Y7I enzyme. Computational studi es of the proton transfer step in catalysis by carbonic anhydrase show that water wires consisting of fewer molecules transfer protons more efficiently ( 1 31, 164 ) Wild type HCA II and Y7I have an identical water structure in the active site cavity with a branched cluster of four ordered water molecules between His64 and the zinc bound water (Figures 4 1, 4 4). In contrast, Y7F has smaller, unbranched clus ter of three water molecules that is more stable and provides a proton transfer pathway of lower energy barrier than wild type ( 164 ) This provides an explanation of why Y7I and wild type, as well as other mutants, lie on the line of Figure 4 11 but Y7F lies above it, other variables being equal. It is interesting to speculate why Tyr7 is conserved when substitution can enhance the rate of maximal catalysis. Calorimetry showed that the replacements of Tyr7 in HCA II decreased the ther mal stability of the protein by 7 10 C, except for Y7F which wa s decreased about 4 C (Table 4 7 ). This decreas ed stabilization indicates that Tyr 7 stabilizes the enzyme, although it is unclear how it does this since the side chain of Tyr7 has no appare nt interactions with other residues in the crystal structure. At any rate, this decreased stabilization may be a factor to explain the occurrence of Tyr7 in many of the
103 terminal chain, which appears to have another role. A conserved N terminus is consistent with this ( 165 ) Recent deletion and mut ation studies have shown the numerous histidine residues (His3, His4, His10, His15 and His 17) found in the N terminus of HCA II represent an acidic motif important for binding the C terminus of AE1 and AE2 ( 165 )
104 Table 4 1 Crystal structure data and refinement statistics of four variants of HCA II N62A N62V N62T N62D Y7I Space Group unit cell dimensions, a, b, c (), ( ) P 2 1 42.8 41.7 72.9 104.6 P 2 1 42.8 41.7 72.9 104.6 P 2 1 42.7 41.6 72.8 104.6 P 2 1 42.7 41.7 72.8 104.5 P 2 1 42.8 41.6 73.2 104. 9 R esolution () 50 1.7 (1.76 1.7) a 50 1.5 (1.55 1.5) 50 1.6 (1.66 1.6) 50 1.6 (1.66 1.6) 20 1.5 (1.55 1.5) R symm b (%) 0.08 0 (0.45) 0.074(0.42) 0.065(0.34 ) 0.054(0.33) 0.099 (0.41) C ompleteness (%) 25.3 (3.1) 93.8(91.5) 31.3 (3.2) 92.2(87.0) 12.9 (3.1) 94.3(90.6) 28.8 (4.6) 95.2(91.1) 23.4 (3.5) 93.0(90.1) Redundancy 2.4(2.3) 2.6(2.5) 2 .9(2.9) 2.9(2.7) 2.7(2.4) No. of unique reflections 25972(2500) a 37008(3472) 31194(2971) 31546(2992) 38859(3606) R cryst c / R free d (%) 17.9 / 19.5 18.4 / 19.7 17.05 / 19.5 17.9 / 18.3 16.6/21.1 No of protein/solvent atoms 2055/110 2057/201 2057/179 2058/28 9 2059/186 average B factors( 2 ) main/side chain Zn/solvent 18.2/21.3 11.9/36.3 15.8/19.0 9.5/35.7 13.1/16.2 6.8/29.3 15.3/18.3 9.3/42.3 17.3/22.7 11.7/30.2 Ramachandran Plot (%). most favored/ additionally allowed, 96.9/3.1 96.9/3.1 96.1/3.9 96.5/3.5 99.5/0.5 R.m.s.d. for bond lengths () / angles ( ) 0.007 / 1.3 0.004 / 1.3 0.004 / 1.3 0.005 / 1.3 0.007/1.1 a Values in parenthesis are f or the highest resolution shell. b R symm c R cryst = 100 d R free is calculated the same as R cryst except it uses 5% of reflection data omitted from refi nement.
105 Table 4 2 Mean B factors ( 2 ) for the ordered side chains of His64 and amino acids in its immediate vicinity in the active site cavity of HCA II and variants. Wild type a N62A N62V N62T N62D His64 13.2 b / 8.5 c 24.9 b 19.6 b 12.5 b / 14.7 c 18.2 c Trp5 11.3 20.4 16.8 14.3 17.2 Tyr7 8.9 14.9 13.0 9.5 12.1 a Data from the Protein Data Bank, accession code 2CBA solved to a comparable resolution of 1.5 b c
106 Table 4 3 Apparent values of pK a obtained by various kinetic measurements of catalysis by HCA II and mutants. Enzyme Orientation of His64 pK a His64 a (eq 6) pK a ZnH2O a (eq 6) pK a ZnH2O b (eq 5) pK a ZnH2 O (esterase) wild type in/out 7.2 6.8 6.9 6.9 N62T in/out 7.0 7.0 7.0 7.0 N62D out 8.4 7.6 7.9 7.7 N62A in 6.2 6.5 7.2 7.1 N62V in 5.9 5.9 7.3 7.1 N62L c in 6.0 d 6.0 d 7.3 7.1 a Measured from the fits of eq 6 to data of Figure 5. The values of p K a have standard errors generally near 0.1 and no greater than 0.2. b Measured from the data of Figure 4 using eq 5. The standard errors in pK a are mostly 0.1 and no greater than 0.2. c These data from Fisher et al. ( 113 ) d These values estimated from poorly resolved pH dependence shown in Fisher et al. ( 113 )
107 Table 4 4 Maximal values of rate constants for hydration of CO 2 for hydrolysis of p nitrophenylacetate and for proton transfer in the dehydration direction catalyzed by HCA II and variants. a Variant of HCA II Orientation of His64 ( k cat ex /K eff CO2 ) b hydration k cat /K m esterase k B b dehyd mM 1 s 1 M 1 s 1 ms 1 wild type in/out 120 2800 0.80 N62T in/out 69 2880 0.39 N62D out 53 1900 0.043 N62A in 81 2220 0.39 N62V in 83 2360 0.35 N62L c in 140 2050 0.20 d a The standard errors for these rate constants are 20% or less. b Measured from the exchange of 18 O between CO 2 and water using eqs 5 and 6. c These data from Fisher et al. ( 11 3 ) d This value estimated from poorly resolved pH dependence shown in Fisher et al. ( 113 )
108 Table 4 5. Maximal Values of Rate Constants for Hydrat ion of CO 2 Hydrolysis of 4 Nitrophenylacetate, and Proton Transfer in Dehydration Catalyzed by HCA II and Variants. a Enzyme k cat exch /K eff CO2 CO 2 hydration 1 s 1 ) b k cat / K m esterase (M 1 s 1 ) c k B proton transfer 1 ) d wld type 120 2800 0.8 Y7I 130 2400 2.3 Y7A 140 2300 0.8 Y7W 140 1700 1.8 Y7F e 120 4400 7.0 Y7D 130 1800 0.8 f Y7N 120 1200 2.5 f Y7R 120 1700 1.5 Y7S 120 1600 1.0 a Derived from the kinetic curves for each substitution by a fit of to the. All data were obtained at 25 o C. The s tandard errors for these rate constants are generally 20% or less. b Measured from the exchange of 18 O between CO 2 and water using eq 5 in the hydration direction. c Measured from the fit of the rate constants for ester hydrolysis to a single ionization. d Measured from the exchange of 18 O between CO 2 and water using eq 6 in the dehydration direction. e Data are from Fisher et al. ( 113 ) f These ar e maximal values of R H2O /[E] since incomplete pH profiles did not allow an adequate determination of k B by a fit of eq 6. Rose Mikulski of the Silverman lab performed these measurements.
109 Table 4 6. Values of Apparent p K a Obtained by Various Kinetic Meas urements of Catalysis by HCA II and Mutants Enzyme p K a His64 a (eq 6) p K a ZnH2O a (eq 6) p K a ZnH2O b (from k cat exch /K eff CO2 ) p K a ZnH2O (esterase) wild type 7.2 6.8 6.9 6.9 Y7I 6.2 6.8 7.1 6.9 Y7A 6.4 6.4 7.0 7.2 Y7W 6.9 7.0 7.2 7.0 Y7F c 6.0 c 7.0 c 7.1 c 7.0 Y7D -d -d 6.4 6.6 Y7N 6.2 6.8 6.8 6.4 Y7R 6.2 6.2 7.4 7.1 Y7S 6.4 6.4 7.0 6.9 a Measured from the fits of eq 6. The values of the p K a ZnH2O have standard errors generally near 0.1 and no greater than 0.2. b Measured from a fit of eq 5. As evident in these Figures, small perturbations were observed that could be fit by including a second ionization; however, these were not included in this Table. The standard errors in p K a are mostly 0.1 and no greater than 0.2. c These data from Fisher et al. ( 113 ) d For Y7D and Y7N the data for R H2O /[E] did not have sufficient bell shape to be adequately fit by eq 6. Rose Mikulski of the Silverman lab performed these measurements.
110 Table 4 7. Thermodynamics of Unfolding of wt and Y7 variants of HCA II Enzyme T m (C) a H m (kcal mol 1 ) a H vH (kcal mol 1 ) b C p (kcal mol 1 K 1 ) c Temperature range (C) a T 1/2 Width at half peak height (C) a wt 59.50.5 240.02.0 218.02.5 0.72 48.0 70.0 3.60.1 Y7I 51.80.5 180.02.0 153.02.0 0.55 40.0 61.0 5.60.1 Y7A 52.50.5 90.02.0 106.01.2 0.27 39.0 61.0 5.60.1 Y7W 52.00.5 86.02.0 113.01.2 0.26 42.0 60.0 5.30.1 Y7F 55.80.5 55.02.0 87.02.0 0.16 32.0 76.0 9.10.1 Y7D 52.00.5 182.02.0 155.01.5 0.56 40.0 60.0 5.00.1 Y7N 51.80.5 132.02.0 131.02.4 0.40 42.0 61.0 5.30.1 Y7R 49.30.5 138.02.0 142.02.0 0.42 41.9 55.3 5.00.1 Y7S 50.40.5 155.02.0 151.02.0 0.48 40.2 54.9 6 .10.1 a Calorimetric parameters determined by DSC. b H vH ) was determined by fitting thermograms to a two state reversible unfolding model. c C p H m) vs melting temperature ( T m ).
111 Figure 4 1. The active site of HCA II from the data of Fisher et al. ( 41 ) The side chain of His64 is shown in both the inward and outward conformations. The red spheres represent oxygen including the oxygens of ordered water molecules numbered W1, W2, W3a, and W3b. Dashed red lines indicate presumed hydrogen bonds. This figure was generated and rendered with PyMOL (www.pymol.org).
112 Figure 4 2. The active site of site specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D HCA II. Residues are labeled and shown as stick models; the zinc atom is depicted as a white sphere and the oxygens of solvent water molecules as red spheres. Note the side chain of His64 is orientated inward for N62A and N62V, both inward and outward for N62T, and outward for N62D HCA II. The F o F c electron density ma p was generated by omitting the residues at position 62, 64, and five water molecules in the active site. The specific mutant. This figure was generated and rendered with PyMOL (www.pymol.o rg). T199 T200 H64 N62A N67 Zn 2+ Y7 W3a W2 W1 ZnOH /H 2 O W3b H96 H94 T199 T200 H64 N62V N67 Zn 2+ Y7 W3a W2 W1 W3b H96 H94 T199 T200 H64 N62D N67 Zn 2+ Y7 W3a W2 W1 W3b H96 H94 ZnOH /H 2 O ZnOH /H 2 O A D B T199 T200 H64 N62T N67 Zn 2+ Y7 W3a W2 W1 W3b H96 H94 ZnOH /H 2 O C
113 Figure 4 3. Structural superposition of the active site of the site specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D with wild type HCA II. Each panel has the wild type HCA II (grey) structurally aligned with the respective mutant. Residues are labeled and shown as stick models, the zinc atom is depicted as a white sphere and the oxygens of solvent water molecules as red s pheres. The structure of wild type HCA II used was from PDB accession number 2CBA ( 51 ) This figure was generated and rendered with PyMOL ( www.pymol.org ). H94 T199 T200 H64 N62A N67 Zn 2+ Y7 W3a W2 W1 ZnOH /H 2 O W3b H96 H94 Q92 T199 T200 H64 N62T N67 Zn 2+ Y7 W3 a W2 W1 ZnOH /H 2 O W3b H96 Q92 H94 T199 T200 H64 N62V N67 Zn 2+ Y7 W3a W2 W1 ZnOH /H 2 O W3b H96 H94 Q92 T199 T200 H64 N62D N67 Zn 2+ Y7 W3a W2 W1 ZnOH /H 2 O W3b H96 Q92 A C B D
114 Figure 4 4 Crystal structures of the active sites of ( left ) Y7I HCA II superposed with wild type HCA II (gray); and ( right ) Y7F HCA II. The structure for Y7F HCA II is from Fisher et al ( 113 ) This figure was generated and rendered with PyMOL ( www.pymol.org ).
115 Figure 4 5. Overall (A) and N terminus (B) of superimposed crystal structures of wild type HCA II and Y7I HCA II. The superimposed enzyme except the N terminus is represented as a surface. The N terminus of (yellow) wild type; and (green) Y7I HCA II is represented as ribbon, while the respective am ino acids at position 7 as sticks. The hydrophobic and hydrophilic regions of the active site are rendered orange and blue respectively. The active site zinc is depicted as a grey sphere. This figure was generated and rendered with PyMOL ( www.pymol.org ).
116 Figure 4 6 The pH profiles for k cat ex /K eff CO2 for the hydration of CO 2 catalyzed by the following variants of HCA II: wild type ( ); N62V ( ); N62A ( ); N62T ( ); and N62D ( ). Data w ere obtained by 18 O exchange between CO 2 and water measured at 25 C in solutions containing 25 mM of all species of CO 2 and at sufficient Na 2 SO 4 to maintain 0.2 M ionic strength. No buffers were added.
1 17 Figure 4 7 The pH profiles of R H2O /[E], the r ate constant for the release of H 2 18 O from the enzyme, catalyzed by these variants of HCA II: wild type ( ); N62V ( ); N62A ( ); N62T ( ); and N62D ( ). Conditions were as described for Figure 4 4.
118 Figure 4 8 The pH pro files for k cat ex /K eff CO2 (M 1 s 1 ) for the hydration of CO 2 catalyzed 18 O exchange between CO 2 and water using solutions at 25 C containing 25 mM of all species of CO 2 and sufficient Na 2 SO 4 to maintain 0.2 M ionic strength. Rose Mikulski of the Silverman lab performed these measurements.
119 Figure 4 9 The pH profiles for R H2O /[E] (s 1 ) the proton transfer dependent rate of release of 18 O labeled water cat t Conditions were as described in Figure 2. Rose Mikulski of the Silverman lab performed these measurements.
120 Figure 4 10 Differential scanning calorimetry profiles of apparent excess specific heat ( C p ) vs temperature for ( red ) Y7I HCA II; (green) Y7F HCA II and ( bl ack ) wild type HCA II.
121 Figure 4 11 Free energy plot of the logarithm of the rate constant for proton transfer k B (s 1 a ZnH2O pK a His64 ) determined from the R H2O /[E ] pH profiles for the wild type and the mutants of HCA II containing replacements filled symbols ); and (o) for H64A HCA II from An et al. ( 158 ) with prot on transfer provided predominantly by derivatives of imidazole and pyridine acting as exogenous proton donors with the solid line a best fit of Marcus Rate Theory.
122 CHAPTER 5 CONCLUSIONS: STRUCTU RE CATALYSIS CORRELATIONS The observed binding site of CO 2 i n the crystal structure aids in the interpretation of the formation and subsequent release of the product, bicarbonate. Following the nucleophilic attack, two mechanisms have been proposed for the subsequent release of the HCO 3 ion based on the theoretic al free energy calculations of CO 2 /HCO 3 intercoversion. The Lipscomb mechanism ( 37 ) propounds a monodentate Zn HCO 3 intermediate wherein a proton rapidly migrates from the original Zn OH to one of the other two oxygen atoms of the H CO 3 ion. The zinc in this mechanism is held in a tetrahedral coordination ( Figure 5 1A ). In contrast, the Lindskog mechanism ( 36 ) proposes a bidentate Zn HCO 3 intermediate that requires one of the two oxygen atoms of the original CO 2 molecule to coordinate directly with zinc resulting in a penta coordinated metal ion held in a trigonal bipyramidal geometry ( Figure 5 1B ). Xue et al. capture d the bicarbonate in the active site of HCAII using x ray crystallography in a T20 0H mutant that displayed a higher affinity for HCO 3 ion than the wild type enzyme (Figure 5 2 ) (34). Least squares superposition of this structure on the wild type HCA II CO 2 bound structure (C RMSD = 0.21 ) shows that the CO 2 substrate molecule exists in the same plane as the Zn HCO 3 product (Figure 5 2 ). From a strictly structural perspective, the pseudo bidentate nature of the captured Zn HCO 3 complex seems to favor the Lindskog hypothesi s. Nevertheless, in both mechanisms the release of the HCO 3 product from the Zn HCO 3 intermediate is associated with the binding of a water molecule to the metal. The appearance of the previously unseen water, W I in close proximity to the zinc was obser ved in the CO 2 complex structures of both the holo and apo enzymes. This water could be either the displaced W DW water,
123 seen prior to CO 2 binding (Figure 5 2, 5 3 due to change in the local electrostatic environm ent. The position of this water with respect to the zinc leads us to suggest that this water may be the best candidate in the aforementioned water associated displacement of pr oduct bicarbonate T he LBHB as revealed by the 0.9 crystal structure may con tribute to the low pK a near 7 for the zinc bound water molecule, the protolysis of which is enhanced using the energy of formation of the LBHB. In this aspect, the role of the LBHB has an analogy with the catalytic mechanism of liver alcohol dehydrogenase in which proton removal from the Zn coordinated alcohol is promoted by forming a LBHB with Ser48 in the reactant state ( 166 ) The alkoxide then undergoes hydride transfer to generate product. In each case, forming the LBHB provides the energy to pump the proton to His64 in HCA II and to His51 in horse liver alcohol dehydrogenase ( 166 ) Weak hydrogen bonds typical of water molecules in solution have a favorable enthalpy of formation near 5 kcal/mol; however, LBHBs can have such enthalpies near 15 25 kcal/mol ( 139 ) This has significance in the catalysis by HCA II since the binding of CO 2 to its catalytically productive binding site displaces the de ep water molecule W DW ( Figure 5 2, 5 3 ) ( 114, 167 ) and thus requires the cleavage of the LBHB contributing to the very weak binding of CO 2 at this site. A binding constant for CO 2 at its catalytic site in HCA II has been estimated at 100 mM measured by infrared spectroscopy ( 168, 169 ) A tight binding of substrate at the reactive site is a disadvantage for catalysis by HCA II; it adversely affects its physiological function which requires it to enhance catalysis for maximum velocity of k cat = 10 6 s 1 and near diffusion controlled levels for for
124 hydration. In arguments eluc idated by Fersht ( 170 ) the tight binding of substrate (without affecting the transition state) lowers the energy level of the substrate enzyme complex thereby increasing the activation energy of k cat By providing a thermodynamic well or pit that accumulates tightly bound subs trate, the rate of catalysis is decreased. For an enzyme that requires rapid catalysis like carbonic anhydrase, it is advantageous for substrate binding to be weak and the active site to remain largely unbound at physiological levels of substrate CO 2 The concentration of CO 2 in plasma for example is near 1 mM, the value of K m for hydration is 10 mM, and the estimated binding constant of CO 2 is 100 mM. It appears that HCA II evolved wea k substrate binding by having to displace the W DW which participates in a LBHB. It is unclear whether these arguments will apply in the dehydration direction as well for which the maximal catalytic rates are slower than in hydration (maximal steady state constants are k cat /K m = 2 x 10 7 M 1 s 1 and k cat 1 ( 142 ) ). Crystal structures of bicarbonate bound at the active site metal, the presumed catalytic site, have been obtained for the mutant of HCA II with Thr200 replaced by His ( 171 ) with Thr199 replaced with Ala ( 172 ) and for HCA II which Zn(II) is replaced by Co(II) ( 173 ) Although the orientation of the bound bicarbonate is somewhat different in each of these examples, in all three cases the binding of bicarbonate displaces the deep water. The binding constant of bicarbonate at the active site of HCA II is es timated near 100 mM by 13 C NMR measurements ( 155 ) with a similar value estimated by inhibition by bicarbonate of the esterase capacity of HCA II ( 174 ) The value of K m for dehydration is 32 mM ( 142 ) and concentrati on of bicarbonate in plasma is near 24 mM. However, the form of the enzyme that is catalytic in the dehydration direction contains the zinc bound
125 water. This configuration is not comparable to a hydrated hydroxide, and a low barrier hydrogen bond will like ly not be found in this case. Hence, at present we cannot make the argument that weak binding of bicarbonate is caused in part by the displacement of the deep water that participates in a LBHB. This effect of the LBHB in catalysis to weaken substrate bindi ng in HCA II is different than the effect shown in examples for which the formation of a LBHB not in the enzyme substrate complex but in the transition state lowers the overall free energy of activation ( 139 ) In that case a weak hydrogen bond for the substrate enzyme complex becomes a LBHB in the transition state, and the energy released enha nces catalysis by lowering the activation barrier for the catalysis. In conclusion, the reflections of this study contribute not only to the current deliberation on the mechanism of HCA II, but also further the general understanding of enzymatic catalysis
126 Figure 5 2 Proposed mechanisms of HCA II catalysis; Lipscom b (A) and Lindsko g (B).
127 Figure 5 1. Active site. Superposition of unbound holo (13), CO 2 bound holo, and bicarbonate bound T200H HCA II (32). The binding modes of both the CO 2 substr ate and HCO 3 product are similar, with the substrate favoring the hydrophobic side (green) and product favoring the hydrophilic side (orange). DW occupies the area between the side chain of Thr200 and CO 2 (W I cyan) upon CO 2 binding. The CO 2 is orientated so the carbon is primed for the nucleophilic attack by the zinc bound hydroxide (orange sphere). A superposition of the V143Y variant of HCA II (9, 27). Note the side chain of Tyr143 (wh ite) acts as a steric block to the CO 2 binding site. Figure created using PyMOL (www.pymol.org).
128 Figure 5 3 Proposed catalytic mechanism of HCA II Schematic representation of three discrete stages of the catalytic cycle. (a) Unbound: note the pres ence of deep water (W DW ); (b) CO 2 bound; note the displacement of W DW and the hydrogen bond between substrate and backbone amide of Thr199; (c) formation of bicarbonate. Figure created using ChemDraw 11.0 (www.cambridgesoft.com).
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144 BIOGRAPHICAL SKETCH Balu earned his Bachelor of Scienc e in 2003 at Andhra Loyola College in Chemistry, Zoology and Botany while loiterin g away most of his free time head-banging to 80s and 90s rock. With a stiff neck he went on to pursue masters degree in Biotechnology at the PSG College of Arts and Science. During his masters work, he interned at the National Instit ute of Immunology, New Delh i, where he researched the phagolysosome modulation Mycobacterium tuberculosis. Balu joined the McKenna lab at the University of Florida in the fall of 2006. His doctoral research was on the structure and catalysis at the active site of human carbonic anhydrase II. Balu likes to paint oil on canvas, party with friends, travel the worl d, skydive, scuba dive, camp, blaze and listen to psychedelic rock.