Structural Modification of Human Carbonic Anhydrase Ii (Hcaii) and Its Impact on Catalysis

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Structural Modification of Human Carbonic Anhydrase Ii (Hcaii) and Its Impact on Catalysis
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West, Dayne M
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Biochemistry and Molecular Biology (IDP)
Committee Chair:
Mckenna, Robert
Committee Co-Chair:
Silverman, David N
Committee Members:
Edison, Arthur S
Frost, Susan C
Kem, William R

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anhydrase -- carbonic -- structure
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
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Abstract:
Human Carbonic Anhydrase II (HCAII) is a zinc metalloenzyme that catalyzes the conversion of carbon dioxide and water to bicarbonate and a proton, a reaction important for pH regulation, maintenance of CO2 levels and various other physiological processes.  The active site of HCAII is composed a hydrophobic region for CO2 binding and conversion, as well as a hydrophilic region for proton transfer.  Using site directed mutagenesis, residues in the active site were altered and structural and kinetic effects were studied.  Mutations to residue V143 to isoleucine in the hydrophobic region of the active site produced a variant with an almost 20 fold decrease in catalytic efficiency.  The structure of this variant was solved with x-ray crystallography and the appearance of product bicarbonate was seen, an amazing occurrence highly uncommon in structural enzymology.  From these data we propose the appearance of bicarbonate is a result of disruption in product dissociation due to steric crowding of the transition state from the larger isoleucine.  Analysis of the hydrophilic region through mutagenesis of N67 and Y7 to glutamine and phenylalanine produced significant structural and catalytic changes.  Kinetic analysis showed that these HCAII mutants have lower pKa values and faster proton transfer rates, specifically with double mutant Y7F/N67Q with a proton transfer rate nearly ten-fold higher than wild type.  These changes were supported structurally when the crystal structure of Y7F/N67Q showed a more linear solvent network through the weakening of hydrogen bonds, the removal of a water molecule from the solvent network, and a shorter distance from His64, the proton shuttle of HCAII.  These results provide data indicating mutations in the active site region that can induce drastic changes in catalytic behavior, allowing the identification and acknowledgement of residues important for maximal activity of HCAII.
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by Dayne M West.
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Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Mckenna, Robert.
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Co-adviser: Silverman, David N.
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1 STRUCTURAL MODIFICATION OF HUMAN CARBONIC ANHYDRASE II (HCAII) AND ITS IMPACT ON CATALYSIS By DAYNE MARCO WEST A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE RE QUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Dayne West

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3 To my family

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4 ACKNOWLEDGMENTS I would first like to thank my Lord and Savior, Jesus Christ, because without him, nothing is possible. I want to especially thank my large and supportive family. My parents have always been proud and supportive of me. I want to thank you both for always being one phone call away and pillars of seemingly infinite knowledge. I would like to thank my grandmother, Se nella, who has been the perfect combination of love and truth, providing wisdom and advice that has significantly helped mold the man I am today. Finally, to all of my aunts, uncles and cousins, your constant support and unconditional love keeps me motiva ted to succeed and make you proud. I would like to thank Dr. Fatma Helmy and the Minority Access to Research Careers (MARC) office at Delaware State University. Being a MARC scholar, I was able to participate in very important research an present my re sults at annual science meetings, where I met recruiters for the University of Florida. I would like to thank Dr. Wayne T. McCormack and the staff in the Biomedical Sciences Office. Dr. McCormack recommended me for the Bridge to Doctorate Fellowship and convinced me that Gainesville was where I belonged. I want to Valerie and Theresa: thanks for all the important little things you have done that made my graduate life much easier to handle. I must also thank Mr. Earl Wade, Dr. Laurence Alexander and Dr. Frierson Being a part of the Bridge to Doctorate program was a fulfilling and enlightening experience that has greatly prepared me for the next phase of my research career. I would like to acknowledge those who have closely helped me in my research. Dr Rose Mikulski, thank you for training and working so closely with me. Your constantly cheerful demeanor pulled me out of multiple emotional downward spirals. To Dr. Kat Sippel, thank you for giving me an example of what a diligent, hard working scienti st is. To Jeanne, thank you for

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5 teaching me to be patient and helping me break some bad research habits. To Dr. Art Robbins, thank you for helping me understand what it means to be a crystallographer; it was a privilege to work with you. To all my lab m ates of the past, present, and future, we are not just a lab, but a family. I want to thank Dr. Chingkuang Tu who has helped me on every project I have been a part of and is someone who I aspire to resemble in the future of my scientific career. I would like to thank all of the friends I have made while attending graduate school. I have so many memories that will stay with me forever. I want to say thanks to Kerri, who introduced me to a world that I never knew existed. I would like to thank the many p rofessors and scientists I have interacted with over the years. I appreciate the time, motivation and wisdom that you all have given me. I would like to thank my committee members for all of their valuable advice and dedication to ensuring my success as a young scientist. I do not believe I could have assembled a better group of scientists. I want to recognize Dr. David Silverman. It has been an absolute honor these four years work side by side with a scientist, and a man of your caliber. Thank you fo r always being there to talk, whether it concerns research, or life. Finally I would like to thank my mentor, Dr. Robert McKenna. I do not believe I will ever have a mentor as amazing as you. The knowledge, time, and effort you have put in to ensure my success is unbelievable. I look at you and see the scientist I want to be. There are not enough words to express how much I appreciate you. Thank you Dr. McKenna, and thank everyone who has made this achievement possible.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Carbonic Anhydrase ................................ ................................ ............................... 15 The CAs ................................ ................................ ................................ ........ 15 CAs ................................ ................................ ................................ ............... 17 The CAs ................................ ................................ ................................ ........ 18 class ................................ ................................ ................................ ..... 19 Structure of HCAII ................................ ................................ ................................ ... 20 CO 2 Binding Pocket ................................ ................................ .......................... 21 Hydrophilic Region/Proton Transfer ................................ ................................ 23 2 METHODS ................................ ................................ ................................ .............. 43 Site directed Mutagenesis/Polymerase Chain Reaction ................................ ........ 43 PCR Process ................................ ................................ ................................ .... 43 PCR Reaction Materials ................................ ................................ ................... 44 DNA Transformation ................................ ................................ ......................... 44 Protein Expression ................................ ................................ ................................ .. 45 Affinity Chromatography/Buffer Exchange ................................ .............................. 46 18 O Mass Spectrometry ................................ ................................ .......................... 47 Stopped flow Spectrophotometry ................................ ................................ ........... 50 X Ray Crystallography ................................ ................................ ........................... 50 Crystals and X rays ................................ ................................ ......................... 51 The Unit Cell ................................ ................................ ................................ ..... 53 Growing Crystals ................................ ................................ .............................. 53 Data Processing ................................ ................................ ............................... 55 Phasing ................................ ................................ ................................ ............ 57 3 CAII AND CYANATE BINDING ................................ ................................ .............. 65 Anion Binding and Metal Substitution in HCAII ................................ ....................... 65 Inhibitor Coordination in Active CAs ................................ ................................ 65 Tetrahedral coordination ................................ ................................ ............ 66

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7 Penta coordination ................................ ................................ .................... 66 Uncoordinated binding ................................ ................................ ............... 67 NMR Experiments Promote Controversy with Cyanate Binding ....................... 67 Structural Data Promotes That Cyanate Is a Zinc bound Ligand ..................... 67 Materials and Methods ................................ ................................ ............................ 68 Expression and Purification of Mutants. ................................ ........................... 68 Crystallization. ................................ ................................ ................................ .. 68 Data Collection ................................ ................................ ................................ 68 Structure Solution and Model Refinement ................................ ....................... 69 Enzyme Titration: Inhibitor Affinity ................................ ................................ ... 69 Results ................................ ................................ ................................ .................... 70 Crystal Structures ................................ ................................ ............................. 70 Inhibitor Affinity ................................ ................................ ................................ 71 Discussion ................................ ................................ ................................ .............. 71 4 CO 2 BINDING AND CATALYTIC EFFICIENCY ................................ ...................... 86 The Hydrophobic Pocket ................................ ................................ ......................... 86 Mate rials and Methods ................................ ................................ ............................ 87 Expression and Purification of Mutants. ................................ ........................... 87 Crystallization. ................................ ................................ ................................ .. 88 CO 2 Binding. ................................ ................................ ................................ ..... 88 Data Collection. ................................ ................................ ................................ 88 Structure Solution and Model Refinement. ................................ ....................... 89 18 O Exchange Mass Spectrometry ................................ ................................ ... 89 Stopped Flow Spectrophotometry ................................ ................................ .... 90 Results ................................ ................................ ................................ .................... 90 Catalysis ................................ ................................ ................................ ........... 90 Crystal Structures. ................................ ................................ ............................ 91 Substrate and Product Binding in V143I HC AII ................................ ................ 92 Discussion ................................ ................................ ................................ .............. 93 5 SOLVENT NETWORK, PROTON TRANSFER AND MARCUS THEORY ........... 110 Methods ................................ ................................ ................................ ................ 111 Site directed Mutagenesis ................................ ................................ ............. 111 18 O Exchange ................................ ................................ ................................ 111 Stopped flow Spectrophotometry ................................ ................................ ... 111 Crystallography ................................ ................................ .............................. 112 Results ................................ ................................ ................................ .................. 113 Catalysis ................................ ................................ ................................ ......... 113 Crystal structures ................................ ................................ ........................... 113 Discussion ................................ ................................ ................................ ............ 116 Solvent Network and Proton Transfer ................................ ............................ 116 Marcus Theory ................................ ................................ ............................... 119

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8 6 FUTURE STUDIES ................................ ................................ ............................... 137 HCAII in Environmental and Artificial Lung Research ................................ ........... 137 HCAII and Proton Transfer ................................ ................................ ................... 138 APPENDIX A SEQUENCE ALIGN MENT OF MAMMALIAN CAS ................................ ............... 141 LIST OF REFERENCES ................................ ................................ ............................. 146 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 156

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9 LIST OF TABLES Table page 1 1 ................................ ................................ .............. 26 1 2 Sequence identity for CAs. Values are in percentage a ................................ ... 27 3 1 Data and refinement statisti cs for cyanate bound to variants of HCAII ............... 75 3 2 Inhibition constants for cyanate bound in HCAII variants a ................................ .. 76 4 1 Data processin g and refinement statistics for the structures of the HCAII variants ................................ ................................ ................................ ............... 98 4 2 Maximal values of rate constants for the hydration of CO 2 and dehydration of bicarbonate catalyzed by variants of HC AII, and related pK a values .................. 99 4 3 Steady state constants for catalysis of the hydration of CO 2 by variants of HCAII obtained by stopped flow spectrophotometry at 25 C and pH 8.3 ........ 100 4 4 Interatomic distances in for CO 2 in HCAII and variants. ................................ 101 5 1 Crystal Structure Data and Refinement Statistics for N67Q and Y7F+ N67Q HCAII ................................ ................................ ................................ ................ 124 5 2 Maximal Values of Rate Constants for Hydration of CO 2 and Proton Transfer in Dehydration Catalyzed by HCAII and Variants ................................ ............. 125 5 3 Values of Apparent pK a Obtained by Kinetic Measurements of Catalysis by HCAII and Variants ................................ ................................ ........................... 126 5 4 Steady state constants for the hydration of CO 2 and dehydration of bicarb onate catalyzed by wild type and Y7F N67Q HCAII a .............................. 127 5 5 Comparison of Distances () in the P roposed hydrogen B ond N etwork .......... 128 5 6 Parameters of the Marcus Equation for Activation of H64G HCAII by Exogenous Proton Donors in H 2 O and D 2 O (98%) a ................................ .......... 131

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10 LIST OF FIGURES Figure page 1 1 ................................ .... 28 1 2 CAs ................................ ................................ ............... 29 1 3 Structure of Human Carbo nic Anhydrase Related Protein (CARP) isoform VIII. ................................ ................................ ................................ ..................... 30 1 4 Structure of the catalytic domain of Human Carbonic Anhydrase IX, an example of a membrane associated isoform of CA. ................................ ........... 31 1 5 A close up of the active site ................................ ................................ ............... 32 1 6 structure of Beta Carbonic Anhydrase Cab from Methanobacterium thermoautotrophicum ................................ ................................ ....................... 3 3 1 7 Close up of the active site CAs from each subclass. ................................ 34 1 8 CA family. ................................ ............. 35 1 9 Active site of Zn and Cobalt substituted Cam. ................................ ................... 36 1 10 Structure of Zn and Cd CA CDCA1 ................................ ............................. 37 1 11 Stick diag ram of the active site of HCAII. ................................ ........................... 38 1 12 Active site of wild type HCAII with substrate CO 2 bound ................................ .... 39 1 13 Theoretica l catalytic mechanism of CAII ................................ ............................. 40 1 14 Branched water network in the active site of wild type HCAII ............................. 41 1 15 Y7F active site structure ................................ ................................ ..................... 42 2 1 Diagram of steps involved in Polymerase Chain Reaction (PCR) ...................... 59 2 2 Site Directed Mutagenesis i nvolves the use of PCR for synthesis of mutant DNA, followed by digestion of nonmutated parental DNA. ................................ 60 2 3 Carbonic Anhydrase catalyzed oxygen 18 exchange at chemical equilibrium. .. 61 2 4 Basic setup of a stopped flow spectrophotometry experiment ........................... 62 2 5 ................................ ................................ ..... 63 2 6 Electron Density Map of V143A ................................ ................................ .......... 64

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11 3 1 H CAII in complex with bromide ................................ ................................ ........... 77 3 2 H CAII in comple x with sulfite ................................ ................................ .............. 78 3 3 H CAII in complex with azide ................................ ................................ ............... 79 3 4 H CAII in complex with formate ................................ ................................ ........... 80 3 5 HCAII in complex with cyanate ................................ ................................ .......... 81 3 6 V207I HCAII in comp lex with cyanate ................................ ................................ 82 3 7 Overlay of wild t ype and V207I HCAII in complex with cyanate ......................... 83 3 8 Overlay of H CAII/anion complexes ................................ ................................ ..... 84 3 9 HCAII with cyanate and carbon dioxide ................................ ............................ 85 4 1 pH profiles of k cat ex /K eff CO2 1 s 1 ) for the hydration of CO 2 catalyzed by variants of HCAII. ................................ ................................ ............................ 102 4 2 pH profiles of RH 2 O/[E] (s 1) for the hydration of CO 2 catalyzed by the following variants of HCAII. ................................ ................................ ............... 103 4 3 Crystal structure at the active site of the V143I HCAII ................................ ..... 104 4 4 Stick overlay of position 143 HCAII structures. ................................ ................. 106 4 5 Stick stereo figure of the active site of V143I HCAII CO 2 /bicarbonate complex.. ................................ ................................ ................................ .......... 107 4 6 Structure of substra te/product bound in HCAII V143I a s observed in the crystal structure ................................ ................................ ............................... 108 4 7 Comparison of HCAII V143I substrate and product binding in HCAII .............. 109 5 1 Structure of the active site of N67Q HCAII ................................ ....................... 129 5 2 Active site structures for Y7FY7F N67Q HCAII crystallized at pH 8.0 .............. 130 5 3 Activation in H 2 O and D 2 O (98%) of R H2O /[E] by 4 methylimidazole in catalysis of 18 O exchange by H64G HCAII. ................................ ...................... 132 5 4 The dependence on pH of the rate constant catalyzed by H64G HCAII in the absence of b uffer and in the presence of 100 mM 4 methylimidazole. ............. 133 5 5 Dependence on pH of k cat ex /K eff S for the hydration of CO 2 catalyzed by ( ) H64G HCAII. ................................ ................................ ................................ .... 134

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12 5 6 Values in H2O and D2O for k B for the activation of catalysis by H64G HCAII versus the difference in pK a values. ................................ ................................ 135 5 7 Values in H 2 O for k B for the activation of catalysis by N62L H64A N67L HCAII vers us the difference in pK a values ................................ ....................... 136 6 1 Overlay of wild type HCAII and HCAIX mimc .. ................................ ............... 140

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13 A bstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURAL MODIFICATION OF HUMAN CARBONIC ANHYDRASE II (HCAII) AND ITS IMPACT ON CATALYSIS By Dayne Marco West August 2012 Chair: Robert McKenna Major: Medical Sciences Biochemistry and Molecular Biology Human Carbonic Anhydrase II (HCAII) is a zinc metalloenzyme that catalyzes the conversion of carbon dioxide and water to bicarbonate and a proton, a reaction important for pH regulation, maintenance of CO 2 levels and various other physiological processes. The active site of HCAII is composed a hydrophobic region for CO 2 binding and conversion, as well as a hydrophilic region for proton trans fer. Using site directed mutagenesis, residues in the active site were altered and structural and kinetic effects were studied. Mutations to residue V143 to isoleucine in the hydrophobic region of the active site produced a variant with an almost 20 fold decrease in catalytic efficiency. The structure of this variant was solved with x ray crystallography and the appearance of product bicarbonate was seen, an amazing occurrence highly uncommon in structural enzymology. From these data we propose the appe arance of bicarbonate is a result of disruption in product dissociation due to steric crowding of the transition state from the larger isoleucine. Analysis of the hydrophilic region through mutagenesis of N67 and Y7 to glutamine and phenylalanine produced significant structural and catalytic changes. Kinetic analysis showed that these HCAII mutants have lower pK a values and faster proton transfer rates specifically with double mutant Y7F/N67Q with a proton

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14 transfer rate nearly ten fold higher than wild t ype These changes were supported structurally when the crystal structure of Y7F/N67Q showed a more linear solvent network through the weakening of hydrogen bonds, the removal of a water molecule from the solvent network, and a shorter distance from His64 the proton shuttle of HCAII. These r esults provide data indicating mutations in the active site region that can induce drastic changes in catalytic behavior, allowing the identification and acknowledgement of residues important for maximal activity of H CAII.

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15 CHAPTER 1 INTRODUCTION Carbonic Anhydrase Carbonic Anhydrases (CAs) are a famil y of enzymes associated with various diseases and physiological functions. The main reaction mechanism involves the highly efficient interconversion of carbon dioxide and water into bicarbonate and protons (1) CAs have been shown to contain a n array of metal ions, like zinc, iron, cobalt and cadmium that are necessary for enzymatic activity ( 2 3, 4 5 ) As a result, CAs are referred to as metalloenzymes, and are exp ressed throughout various species. There are 5 structurally unrelated classes of CA ( 1 7 ) The CAs primarily in mammals (Figure 1 1) There are 16 isoforms of CA in this family, and are expressed in various tissues (Table 1 1) The active isoforms in this family contain a zinc atom necessary for catalytic activity coordinated by three histidines and a water/hydroxide molecule The enzymes of this class are localized throughout the cell ( 6 ). Members that are c ytosolic include CA isoforms I III, VII, VIII, X, XI and III (Figure 1 2) S ome members of this class are expressed in the mitochondria (CAV A&B), or mem brane associated (IV, IX, XII, XIV and XV (in mice) ). While CA IV is anchored to the membrane by a glycophosphatidylinositol ( GP I ) tail, isoforms IX (Figure 1 4), XII, and XIV are transmembrane proteins that contain an N terminal extracellular catalytic (CA) domain, a helical domain that spans the mem brane, and an intracellular C terminal domain ( 7 8 ). There is also CA VI that is a secretory isofo rm expressed in saliva and mammary glands The various isoforms vary in structure as well as activity. Some isoforms, for

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16 example CAII and CA IV are very active, with turnover rates of 10 6 s 1 However, isoforms VIII, X, and XI are catalytically inactiv e, a result of these isoforms lacking zinc in the active site (6 9, 1 1 ) ( Figure 1 3) The se CA related proteins (CARPs) lack one or more histidine residues to stabilize the ion. CARPs are expressed primarily in the central nervous system ( 6 12 ) Asi de from CO 2 hydration, t his class of CAs has also been heavily associated with important chemical reactions such as cyanate hydration, aldehyde hydration and hydrolysis ( 1 3 ) The class of CA has been implicated in many important physiological functions. CAII, the most abundant and most active isoform, is ubiquitously expressed throughout various cells in the body including cells that make up bone, brain, kidney, eye, liver and blood ( 14 15 ) One of the most important f unctions of CA is the assist ance i n acid base homeostasis This involves CO 2 and bicarbonate transport between tissues as well as excretion and removal CA related processes involving isozymes I, II and IV include facilitated CO 2 removal in the pulmonary vasculature of lungs, eliminatio n of protons and reabsorption of bicarbonate by the kidneys ( 1 7 ) and acidification of the bone resorbing compartment by osteoclasts ( 16 ) Medications for glaucoma are typically inhibitors of CAII, as they inhibit CAII from secreting bicarbonate, lowering intraocular pressure of the bicarbonate rich aqueous humour within the eye ( 10 ) Other functions of CA include cerebrospinal fluid formation, saliva production, gastric acid production, intestinal ion transport, gustation and some muscle function ( 6 1 7 ) Evidence of the importance of CAII is further implicated by CAII deficiency syndrome, a disease in which the body does not actively express CAII. People affected with this disease have suffered from renal

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17 tubular acidosis, cerebral calcification and o steopetrosis Some isozymes, like CA IX, XII, and VIII are highly expressed in tumors, involved in tumor progression (16, 18, 19 ) The reaction catalyzed by active CAs is a two stage ping pong mechanism of the reversible hydration of CO 2 to bicarbonate and a proton. 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 hydro xide. The resultant bicarbonate is then displaced from the zinc by a water molecule (eq 1 1 ). To regenerate the zinc bound hydroxide, the active form of the enzyme, a proton transfer reaction occurs (eq 1 2) between the zinc bound water and external buffer (B) or solvent The transfer of protons in and out of the active site is typically assisted by a proton shuttle such as His64 in H CAII ( 20, 21 ) H 2 O CO 2 + EZnOH EZnHCO 3 EZnH 2 O + HCO 3 (1 1 ) EZnH 2 O + B EZnOH + BH + ( 1 2 ) CAs Beta CAs are expressed in prokaryotes, as well as chloroplasts in plants. In chloroplasts, CA assist s in the regulation of CO 2 concentration which is im portant for ph ysiological functions in plants ( 22 ) One important role is that of photosynthesis, in which CA operates in numerous roles. The rapid interconversion of HCO 3 and CO 2 by CA permits efficient fixation for Rubisco and P hosphoenylpyruvate (PEP) carboxylase Rubisco converts CO 2 to a usable energy source in plants, and PEP carboxylase catalyzes the addition of HCO 3 to PEP. Both carbon fixation reactions are important for photosynthesis, and the two substrates, CO 2 and bicarbonate, are produc ts of CA CA a lso plays a role in CO 2 uptake in aquatic phototrophs, as well as a specialized role of facilitated diffusion of CO 2 ( 22 ) This class of enzymes is also seen as a target for drug

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18 design to prevent infection from pathogenic species of bacteria like Helicob acter pylori and Mycobacterium tuberculosis ( 1 7 ) The class of CA is multimeric and show wide structural variations (Figure 1 6) ( 23 ) Structures of this class of enzymes indicate two further sub classes In one class, t he zinc in this class is coordinated with a histidine residue, two cysteine residues, and the fourth coordination site is occupied by a water /hydroxide molecule similar to the class This is seen in enzymes from Pisum sativum, M ethano bacterium t hermoautotrophicum and several others (Figure 1 7 A) ( 24 26 ) However, CAs from other spe cies, such as Porphyridium purpureum and Escherichia coli the zinc is coordinated by t wo cysteine residues histidine and an aspartate ( 27 28 ) At pH lower than 7.5, the aspartate binds to the zinc as a fourth ligand, preventing solvation (Figure 1 7 B) ( 1 7 ). It has been shown that at pH above 8.3, the binding aspartate forms a salt bridge with a conserved arginine residue, allowing access of a hydroxide/water molecule to the coordinate metal. This generates the tetrahedral geometry similar to the class. The proposed catalytic mechanism for the class is identical to the class, although the proton transfer step i s currently not well understood ( 29 ) The activity for this enzyme appears to be significantly lower in comparison to faster members of the class with Cab having a reaction rate of 10 4 s 1 ( 30 ) This is seen in other members of the class ( 23 ). The CAs The gamma class pertains to those expressed in bacteria and archaea ( 30 31 ) An example of this class of enzymes is Cam, a gamma class carbonic anhydrase isolated from Methanosarcina thermophila Cam operates as a homotrimer ( 31 ) The monomers fold in a left helix ( 32 ) In each active site, zinc is coordinated by

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19 two monomers: two histidines from one subunit, a th ird histidine from another and multiple water molecules (Figure 1 8) ( 1 7 ) Metal bindi ng properties also distinguish this class. The metal atom has an additional water ligand, resulting in trigonal bipyramidal coordination geometry, different from the wel l known tertrahedral coordination (Figure 1 9A) The coordination of ligands changes when zinc is substituted with cobalt, from two bound water molecules to three, giving cobalt Cam an octahedral geometry around the metal (Figure 1 9B) ( 33 ). In some cas es, the zinc atom can also be replaced with iron. Studies have shown in similar species that the substitution of iron for zinc yields an enzyme with 3 fold greater catalytic activity ( 34 ). Mechanistically, Cam is proposed to operate in a similar fashion to the class While the active site shows multiple bound solvent molecules, studies indicate that the metal is coordinated by a water molecule and a hydroxide ion, and the hydroxide ion is the only one involved in catalysis One distinguishing occurrence is t hat proton transfer is not accomplished by a histidine, but by two glutamines ( 33, 35 ) Kinetic analysis ly, with a turnover rate of 10 4 s 1 ( 33 ). class There is still much informatio n to be learned from these groups. These classes were discovered in marine diatoms a group of algae and dominant autotroph in marine environments An enzyme known as TWCA1, representing the species from which is isolated, represents the class. CDCA1 represents the class, and uses cadmium as the central metal. Sequence alignment of TWCA1 and structural homology of CDCA1 with other CA families shows few similarities. However, s imilar to the class, these

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20 classes work to produce CO 2 and HCO 3 for ca rbon fixat ion and photosynthesis ( 36, 37 ). These classes also show ambiguity in regards to metal binding. Studies have shown that TWCA1 maintains activity with zinc, or the substitution of cobalt. This is considered an evolutionary tactic due to concentr ations around 2 pM of trace metals in certain parts of the ocean inhabited by diatoms ( 36 ) In regards to CDCA1, it has the ability to equally use and exchange zinc and cadmium (Figure 1 10) The activity of CDCA1 is very high. With a k cat /K m of 8.7x10 8 M 1 s 1 the catalytic efficiency closely approaches the diffusion limit of 10 9 M 1 s 1 Although having low sequence homology, CDCA1 superimposes over the CA dimer from Pisum sativum with a r.m.s.d. of 1.93 over 102 C atoms ( 37 ). Structure of HCAII HC A II is a monomeric metalloenzyme 260 residues in length. The dimensions of the enzyme are approximately 5x4x4 nm 3 The active site is a conical cleft 15 deep with a zinc atom in the interior. The metal is tetrahedral, coordinated by three histidine residues (His94, His96, His119) and a water/hydroxyl group ( 1, 19 38 ) The H CAII active site is partitioned into two distinct ly different surfaces on each side of the zinc ion. On one side is a surface of nonpolar amino acids that form the hydrophobic pocket in which CO 2 positions for the hydration step. Structural studies have mapped out the active site of H CAII and identified a water molecule in the hydrophobic pocket 2.4 away from the zinc bound hydroxide. This water molecule, termed deep water is displaced upon binding of CO 2 ( 38 ) implicating a placeholder role

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21 The other side of the active site is a region lined with hydrophilic amino acids primarily resp onsible for forming the solvent network regulating the proton transfer step (Figure 1 11) Additional structural studies have shown that the hydrophilic portion of the active site contains a well ordered, hydrogen bonded solvent network. This network inc ludes five water molecules. This network allows for movement of protons from the zinc bound solvent to W2, placing it in close proximity to His64 for fast proton transfer ( 39 97,102 ). CO 2 Binding Pocket The CO 2 binding pocket is composed of residues Val1 21, Va1143, Leu198, Val207, the amide group of Thr199 and Trp209 Figure 1 12 shows the substrate binding pocket as well as a secondary CO 2 binding site discussed later. The arrangement of CO 2 in hydrophobic pocket is such that it places the central car bon at a distance of 2.8 from the zinc bound hydroxide This position permits the nucleophilic attack by the lone pair electrons of the zinc bound hydroxyl oxygen (Figure 1 11) (1, 39 ) To this point, methods for accurate kinetic measurements of CO 2 hy dration reactions are not available. However, much work has been done to develop theories on the role of binding pocket residues. Studies have shown that position 143 is very sensitive to changes in activity. When valine is mutated to a bulky residue, such as phenylalanine or tryptophan, CO 2 hydration decreases approximately 3000 fold ( 40 ) It is theorized that these mutations introduce a bulky side chain that protrudes into the hydrophobic cavity, inhibiting prop er substrate binding. Smaller yet stil l significant c hanges also appear to provide catalytic disturbance. The V143I mutation shows a decrease in catalytic efficiency approximately 17 fold. The reasons for this change are harder to deduce, as the increase in molecular size is likely not signi ficant, ho wever structural analysis by x ray crystallography has

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22 shown that there may be a decrease in product dissociation, likely the result of isoleucine crowding an unstable transition state. Simil ar tendencies are seen in Leu143 implicating importa nt roles for valines in CO 2 conversion ( 41 ) Another residue shown to promo te changes in activity is T 199 Work by Liang show s reduction in CO 2 hydration and bicarbonate dehydration to ~1% the activity of wild type when T199 is mutated to alanine ( 3 ) When ions bind to the zinc atom, they donate a hydrogen bond to the hydroxyl of T199. This is a result of Glu106 accepting a hydrogen bond from the hydroxyl of T199. These two residues together are known as the doorkeeper, a bonding network to promote st able complex formation ( 3 ). Proper tetrahedral coordination exhib ited by zinc bound water/hydroxide is accomplished by the stability of its hydrogen bond with T199 X ray crystallography has been very helpful in the analysis of ligand binding and theoreti cal determinations of binding properties. T he structure of HCAII with CO 2 bound in the active site has been solved structurally to high resolution CO 2 binds approximately 2.8 away from the zinc bound water /hydroxide In wild type, this linear molecul from the solvent. T he unshared electrons are in an optimal position to react with the CO 2 produ cing zinc bound bicarbonate (38 41 ) Structures of substrate bound HCAII also ve rify other previous notions. Merz et al predicted using molecular dynamics that there was a second CO 2 binding site ( 42 ). Crystal structures of these complexes show that there is a second CO 2 binding site This site is not considered a second active sit e, but possibly a strong hydrophobic region that can behave like a vacuum and pull in CO 2 especially under high pressure

PAGE 23

23 conditions. This second site is also significantly further away from the active CO 2 binding site, with no current implications on CO 2 hydration ( 38, 42 ) While there is significant structural data regarding CO 2 binding in HCAII, a similar claim can be made for bicarbonate. S tructures of HCAII/HCO 3 complexes are also available and provide interesting structural insight. S tructure s of bicarbonate bound HCAII confirms experimental and theoretical results that bicarbonate binds directly to zinc, implying a conversion of CO 2 and zinc bound hydroxide to zinc bound bicarbonate ( 43 ) Hydrophilic Region/Proton Transfer Carbonic anhydras e II, along with a hydrophobic region, also contains several polar molecules in its active site. These residues are Thr199, Thr200, Tyr7, Asn62, Asn67, and His64, and they make up the hydrophilic region of the active site. Each residue is in proximity to form hydrogen bonds with ordered water molecules that are also present in the active site (101) Thr199 is bonded to the zinc bound solvent, and this interaction stabilizes the solvent and orients the free electrons on the oxygen in a position optimal for reaction with CO 2 ( 44 ) The zinc bound solvent is also hydrogen bonded to another water, W1, which forms a hydrogen bond with Thr200, and a second water in the solvent chain, W2. W2 hydrogen bonds to two branched water molecules, W3A and W3B, each of which interact with Tyr7 ( W3 A) and Asn62 and Asn67 ( W3B interacts with both) via hydrogen bond (Figure 1 14) Various MD simulations plotting solvent occurrences in the active site of HCAII indicated four regions with substantially higher occupancies tha n other waters. These regions correlated with the location of the zinc bound solvent, W1, W3a and W3b ( 45 ) This network is conserved over a wide pH range, with waters W2, W3A and W3B located 2.6 to 3.2 from His64 ( 39 ) The

PAGE 24

24 distance between the zinc b ound solvent and His64 is ~8 which makes direct transfer unfavorable. This provides merit for the idea of a water network facilitating the movement of protons out of the active site. A hypothesis has been developed to incorporate the water network into the proton transfer mechanism It proposes that the proton travel s via the water network using the Grotthuss mechanism, in which protons descend a hydrogen bond network of solvent molecules through covalent bonds ( 46 ). It has been postulated that the pr oton travels down the solvent network forming hydronium ions, until it reaches W2 which is in close enough proximity to transfer to His64. The proton then proceeds to bind to ND1 on the imidazole ring of His64 and is released when the ring flips outward e xposing it to the bulk solvent. This theory is shown as a diagram in Figure 1 13 A major discovery in regards to proton transfer and an associated solvent network arose from the development of Y7F HCAII mutant in which Tyr7 is mutated to phenylalanine Kinetics showed a 7 fold increase in intramolecular proton transfer. The x ray crystal structure shows that the Y7F mutant loses W3A from the network, likely because of the loss of a hydroxyl group preventing hydrogen b ond formation, shown in Figure 1 1 5 A very strong hypothesis is that removal of the branched waters in the solvent network increases proton transfer. Studies have shown that an unbranched water network increases transfer as a result of loss of waters prevent ing formation of H 9 O 4 + a com plex involving W2, W3A and W3B known as an Eigen cation (Figure 1 1 4 ) Although a faster enzyme, the Y7F mutant is less stable, providing an expla n ation why this enzyme is never seen in nature ( 1, 39 ).

PAGE 25

25 Kinetic assays and site directed mutagenesis in CAII has shown that the removal of His64 results in a 20 50 fold decrease in CA activity ( 47 ) The structure of H CAII has been solved and illustrates that His64 has dual conformations directing the indole ring into and out of the active site, strengthening th e hypothesis that His64 flips in to accept a proton, and out to release to bulk solvent ( 45 ) Proton transfer is rapid due to zinc bound hydroxide and His64 each having pK a values around 7. Studies show however that removal of His 64 does not completely inhibit proton transfer, indicating another possible mechanism for this reaction ( 48, 49 ) It has been shown by Duda et al. that proton transfer can be rescued in mutants lacking His64 by v arious pyrimidine buffers ( 50 )

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26 Table 1 1 Isoform k B (s 1 ) k cat /K m (M 1 s 1 ) Cellular Localization Tissue expression HCAI H CAII H CAII I H CAIV HCA VA HCAVB HCAVI HCA VII HCA VIII HCAIX HCAX HCA XI HCA XII HCA XIII HCA XIV mCAXV 0.2 1.4 0.01 1.1 0.3 1.0 0.3 1 0.4 0.4 0.2 0.3 0.5 50 120 0.3 51 29 98 49 83 55 35 11 39 33 Cytosol Cytosol Cytosol Membrane Mitochondria Mitochondria Saliva Cytosol Cytosol Membrane Cytosol Cytosol Membrane Cytosol Membrane membrane Erythrocytes, GI tract Erythrocytes, eye, bone, Skelet al, adipocytes Kidney, lung, colon, heart Liver Cardiac, skeletal, pancreas Salivary, mammary glands CNS CNS Tumors, GI tract CNS CNS tumor, intestine, reproductive Kidney, brain, lung, gut, Kidney, brain, liver Kidney

PAGE 27

27 Table 1 2. Sequence identity for CAs. Values are in percentage a CA 1 2 3 4 5A 5B 6 7 8 9 10 11 12 13 14 15 1 61 54 29 48 48 32 51 38 30 28 26 34 59 33 31 2 58 31 54 53 32 56 38 31 30 31 33 60 34 32 3 30 46 44 30 50 34 30 27 28 33 59 32 29 4 31 30 34 31 27 31 24 22 35 30 3 5 35 5A 65 31 52 38 32 29 31 33 50 32 30 5B 26 47 35 30 26 26 30 49 27 28 6 33 29 39 21 21 38 32 35 31 7 36 33 27 26 36 53 32 31 8 31 26 28 29 36 25 28 9 25 25 38 32 42 34 10 52 27 28 22 21 11 25 29 24 22 12 35 46 35 13 35 33 14 31 15 a Numbers indicate specific isoform of CA. Isoform 15 is mCA15, expressed in mice.

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28 Figure 1 1 Full structure of HCAII, a member of the inc atom is represented by a gra y sphere 15 in the interior ; PDB ID: 2ILI (70) Figure was made using PyMol

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29 Figure 1 CAs HCAI ( blue ), HCAII ( salmon PDB ID: 2ILI ), HCAIII ( red PDB ID: 1Z93 ), HCAVII (green PDB ID: 3MDZ ), HCAVIII (pink PDB ID: 2W2J ), and HCAXIII ( yellow PDB ID: 3D0N ) The members of the family share a mixed / structural motif. R.M.S.D. values range between 0.8 and 1.5 The strong overlay indicates high structural similarit y among the cytosolic members. Figure was made using PyMol

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30 Figure 1 3. Structure of Human Carbonic Anhydrase Related Protein (CARP) isoform VIII. This member of the family is inactive due to lack of zinc. His94 is replaced by Arg116 and Ile224 red uces pocket volume, preventing the binding of zinc or substrate; PDB ID: 2W2J (12) Figure was made using PyMol

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31 Figure 1 4. Structure of the catalytic domain of Human Carbonic Anhydrase IX, an example of a membrane associated isoform of CA; PDB ID: 3IA I. Figure was made using PyMol

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32 Figure 1 5 A close up of the active site shows three histidines that help coordinate the zinc atom ( gray sphere) The active members of the family maintain the zinc atom in the active site through tetrahedral coordination involving the zinc bound solvent and three histidines residues illustrated ; PDB ID: 3IAI. Figure was made using PyMol

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33 Figure 1 6. An example of a CA. S tructure of Be ta Carbonic Anhydrase Cab from Methanobacterium thermoautotrophicum This enzyme is composed of three dimers, with zinc atom indicated by gray spheres; PDB ID: 1G5C (26) Figure was made using PyMol

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34 Figure 1 7. Two types of zinc binding sites are fo und in class CAs. Close up of the CAs from each subclass. A ctive site of Cab (PDB ID: 1G5C) with a tetrahedrally coordinated zinc atom (pink) A ctive site of CA from Porphyridium Purpureum (PDB ID:1DDZ) with aspartate in place of water/hydrox ide (blue) (26, 27) Figure was made using PyMol

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35 Figure 1 CA family This enzyme acts as a homotrimer, indicated by three different colored monomers. Two monomers are required for coordination of the metal ion ; PDB ID: 1THJ (31) The zinc atom is indicated by gray sphere. Figure was made using PyMol

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36 Figure 1 9. Active site of Zn (A) and Cobalt substituted (B) Cam; PDB ID for A: 1QRG, PDB ID for B: 1QQ0 (33) Figure was made using PyMol

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37 Figure 1 10. Str ucture of CA CDCA1. C) Overlay of the two st ructures give a r.m.s.d. of 0.2 PDB ID for A: 3BOC, PDB ID for B: 3BOB ( 5, 36) Figure was made using PyMol

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38 Figure 1 11. Stick diagram of the active site of H CAII Substrate CO 2 (orange), a ctive site solvent (red spheres), hydrophobic pocket (light gray), the hydrophilic region (blue), and the three histidines (yellow) stabilizing the zinc (dark gray). PDB ID: 2ILI and 3D92 (63, 70 ) Amino acids are as labeled. Figure was made using PyMol

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39 Figure 1 12. Active site of wild type HCAII with substrate CO 2 bound (PDB ID: 3D92). The residues in green indicate the substrate binding po cket, and the residues in yellow indicate the second binding site for CO 2 ( 38 ) Figure was made using PyMol

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40 Figure 1 13. Theoretical catalytic mechanism of CAII. Hydrogens are represented by white circles, small gray spheres indicate water molecules in the solvent network, and a large sphere represents the zinc atom. Red arrows indicate passage down the network toward the proton shuttle (H64), indicated as a pentameric ring with in and out conformations (70)

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41 Figure 1 14. Branched water network in t he active site of wild type HCAII. The water molecules are represented by red spheres. The dashed lines indicate distances in the range of hydrogen bond interactions (2.6 to 3.5 ) between waters forming the theoretical solvent network for proton transfe r. Water molecules W1, W2, W3A and W3B form the proposed Eigen Cation complex (1); PDB ID: 2ILI (70) Figure was made using PyMol

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42 Figure 1 15. Y7F active site structure. There is a loss of W3A resulting from the loss of a hydrogen bond acceptor, Y7; P DB ID: 2NXT (71) Figure was made using PyMol

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43 CHAPTER 2 METHODS This chapter provides detail s of the methods used for the presented doctoral work. The mutagenesis w ere performed in conjunction with Polymerase Chain Reaction (PCR). The kinetic data w er e obtained using the Stopped Flow Spectrophotometry a nd Mass Spectrometry Finally, all structural work pres ented here w ere the result of X ray Crystallography. Site directed M utagenesis/Polymerase Chain Reaction Polymerase Chain Reaction (PCR) is a method of analyzing and amplifying short sequences of DNA. Amplification of DNA sections involves the use of primers, two small single stranded DNA sequences synthesized with the purpose of binding to the DNA section of interest. This reaction behaves s imilar to those seen in nature involving DNA replication. The reaction takes place in a thermal cycler, a machine that quickly cycles through various temperatures for varying stages of the process ( 39, 51 ) PCR Process The first stage of the process is d enaturation. In this stage, the cyc ler reaches a temperature of 94C The high temperature promotes separation of hydrogen bonds and denaturation of DNA. This is a requirement for the next stage of the reaction and runs for 30 seconds according to the A gilent Technologies QuikChange Mutagenesis Kit. Following denaturation, th e temperature is lowered to ~54C for 1 minute and annealing occurs. In this step, the formerly denatured strands renature with primers. In a mutagenesis reaction, the primers ar e synthesized with an altered sequence that if successful will bind to the native DNA and induce a mutation in the DNA target sequence. The number of changes to the native sequence increases binding and

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44 annealing difficulty. Following proper annealing o f primers, the final step of the reaction is elongation. In this step, the temperature is raised to 72C an optimal reaction temperature for DNA polymerase. The enzyme then extends the primer sequence to generate a new double stranded DNA molecule. Thi s step goes for 5 minutes and finalizes the first cycle and introduces a newly mutated DNA sequence. This process is recycled to amplify the DNA section of interest (Figure 2 1). Following the cycles, the PCR reaction is then incubated with DpnI, an enzy me that digests native DNA (Figure 2 2) ( 53 56 ) All mutants were developed using the QuickChange site directed mutagenesis kit with reaction mixture listed below. PCR Reaction M aterials 5 l of 10 reaction buffer X l (5 50 ng) of dsDNA template X l ( 125 ng) of oligonucleotide primer #1 X l (125 ng) of oligonucleotide primer #2 1 l of dNTP mix ddH2O to a final volume of 50 l 1 l of Pfu Ultra HF DNA polymerase (2.5 U/ l) DNA Transformation Following successful mutagenesis, the PCR reaction contain ing newly synthesized DNA is transformed into cells. For this work two cell lines were used: XL1 with the cells, and allowing incubation on ice for about 30 min. This s tep stiffens the pores of the cell. Following incubation, cells are heat shocked in a 42 C water bath for 45 seconds to open the pores and permit entrance of DNA into the cell. The cells are next placed on ice for 5 min to close the pores. To promote ce ll growth, media is added to the cell mix and incubated at 37C for 1 hour The final step is the spreading of the

PAGE 45

45 cell/media solution on ampicillin agar plates and incubated overnight at 37 C Successful transformation results in the appearance of cell colonies on the plates. Cell colonies are then picked and mixed with LB media and incubated overnight with agitation (a shaking incubator) to promote cell growth. Working with ampicillin resistant plasmids, ampicillin at a concentration of 100 mg/mL is a dded to prevent growth of unwanted bacteria. DNA was extracted using Eppendorf and Prime plasmid miniprep kits (54) and sequenced by the UF Interdisciplinary Center for Biotechnology Research Protein Expression To obtain enough protein for kinetic and s tructural studies, high volume protein expression is performed. This occurs through the growth of cell cultures in 2 L flasks. The cell line used for protein expression is the BL2l DE3 pLys cell line, a cell line optimized for protein expression and effi cient cell lysis. DNA is transformed into this cell line, and colonies are picked and allowed to inoculate, or grow overnight in a small flask containing 25 to 50 mL LB media Following successful growth, analyzed by the cloudiness of the flask, the cont ents of the small flask are added to a larger 2 L flask containing 1 L of LB media. The most common media used was LB Miller Broth, however in cases of cell growth hindrance, enriched 2XYT media was used. The cells were allowed to grow in the large flask s at 37 C in a shaking incubator until the measured absorbance of the flask contents reached a value of between 0.6 and 1.0 optical density (O.D.) units. This level ensures maximal growth of cells promoting larger production of protein. Once this level h as been reached, protein expression is induced with D 1 thiogalactopyranoside (IPTG), a compound that mimics allolactose inducing transcription of genes in the lac operon. With DNA transformed into this cell line, the transcription machinery c an be used for the gene of interest. The cell s are

PAGE 46

46 allowed to incubate at 37C with agitation. The cells are finally spun down at 5000 rpm to pellet the cells. The supernatant, or used media, is discarded and the pellets are stored at 8 0C To extract protein, frozen pellets are mixed with buffer, lysozyme for cell lysis and deoxyribonuclease for non transcribed DNA degradation ( 55 ) Affinity Chromatography/Buffer Exchange Protein purification from cell lysate is achieved through affinity chromatogra phy. Affinity Chromatography is a method of separating substances based on their affinity to the stationary phase. Columns used were made 2 mL high, composed of a sulfonamide resin that binds to the active site of HCAII. Columns are equilibrated with b uffer containing sodium sulfate and T ris at pH 9 before lysate is added. Prior to purification, the cell lysate is spun down at high speed and the supernatant is collected for purification. After equilibration, supernatant is added to the column and allo wed to pass through the resin, collecting HCAII. The column is then washed with the same equilibration buffer to clean the column and remove excess debris. To ensure all debris removed, the same buffer at pH 7 is applied to the column. For elution of pr otein from the column, 0.4 M s odium azide is used. Sodium a zide binds to the active site stronger than the sulfonamide in the column. Sodium azide is applied to the column, and the eluent, or flow through contains the purified protein. However, sodium azide is an inhibitor of HCAII, so protein analysis would be impossible. As a result, buffer exchange is employed to remove sodium azide and replace it with tris pH 8. Buffer exchange was performed in a Milipore filtration conical vial with a 10 kDa mole cular weight cutoff to prevent HCAII, with a molecular weight of 30 kDa, from flowing through. After filtration with 10 times the eluent volume, the purified sample is concentrated and ready for kinetics and structure work ( 38, 55 )

PAGE 47

47 18 O Mass Spectrometry The origin of the 18 O exchange method was the determination of the rate constant for the uncatalyzed reaction of CO 2 with water. Oxygen 18 exchange is caused by the hydration dehydration cycle at chemical equilibrium. When H 2 16 O is produced by these reac tions, it is essentially infinitely diluted in H 2 16 O of solvent. The measured variable is the rate of decrease in the 18 O content of CO 2 in solution, which approaches with time a value close to the natural abundance of 18 O 0.2%. The rate constant k measur ed by this method remains the best to date for the hydration of CO 2 in acidic solution. The catalysis of this reaction by human carbonic anhydrase II appears to obey Michaelis Menten kinetics under conditions of ample ionic strength and buffering. The ex change of 18 O between species of CO 2 and water is caused by the hydr ation dehydration cycle. The 18 O is exchanged with and nearly infinitely diluted by 16 0 of water; that is, once 16 O appears in solvent water, it is very unlikely to react with CO 2 The ki netic equations for the uncatalyzed depletion of 18 O from labeled CO 2 species in a homogenous aqueous phase were derived by Mills and Urey (Figure 2 3). Experiments with HCAII The 18 O exchange method relies on the depletion of 18 O from CO 2 as measured by membrane inlet mass spectrometry using an Extrel EXM 200 mass spectrometer (17). In the first stage of catalysis, the dehydration of labeled bicarbonate has a probability of labeling the active site with 18 O (eq 2 1 ). In a following step, protonation of th e zinc bound 18 O labeled hydroxide results in the release of H 2 18 O to the solvent where it is very greatly diluted by H 2 16 O (eq 2 2 ).

PAGE 48

48 H 2 O HCOO 18 O + EZnH 2 O EZnHCOO 18 O COO + EZn 18 OH (2 1 ) H 2 O H + His64 EZn 18 OH His 64 EZnH 2 18 EZnH 2 O + H 2 18 (2 2 ) This approach yields two rates: The R1, the rate of CO 2 and HCO 3 interconversion at chemical equilibrium (eq 2 1 ), as shown in eq 2 3 and RH 2 O, the rate of release from the enzyme of water with labeled substrat e oxygen (eq 2 2 ). R1/[E] = k cat exch [CO 2 ]/( K eff CO 2 + [CO 2 ]) ( 2 3 ) In eq 5, k cat exch is a rate constant for maximal interconversion of CO 2 and HCO 3 K eff CO 2 represents a binding constant for the substrate to enzyme. The ratio k cat exch /K eff CO 2 i s considered equivalent in value to k cat /K m from steady state experiments, and is a measure of the successful binding and interconversion of substrate and product. The second rate, RH 2 O, is the component of the 18 O exchange that is dependent upon the dona tion of protons to the 18O labeled zinc bound hydroxide. In such a step, His64 is a predominant proton donor (eq 2 2 ) and the value of RH 2 O can be determined as the rate constant for proton transfer from His64 to the zinc bound hydroxide (48) according eq 2 4 Here k B is the rate constant for proton transfer to the zinc bound hydroxide and (Ka)donor and (Ka)ZnH 2 O are ionization constants of the proton donor, His64, and zinc bound water. The least squares determination of kinetic constants of eqs 2 3 and 2 4 was carried out by using Enzfitter (Biosoft). RH 2 O/[E] = k B /([1 + (Ka)donor /[H + ]][1 + [H + ]/(Ka)ZnH 2 O]) ( 2 4 )

PAGE 49

49 The uncatalyzed and carbonic anhydrase catalyzed exchanges of 18O between CO 2 and water at chemical equilibrium were measured in the absence o f buffer (to prevent interference from intermolecular proton transfer reaction) at a total substrate concentration (all species of CO 2 ) of 25 mM and 25 C. The pH dependence of RH 2 O/[E] at saturating levels of exogenous proton donor is often bell shaped co nsistent with the transfer of a proton from a single predominant donor, in this case an exogenous donor, to the zinc bound hydroxide in the dehydration direction. In these cases the pH profile is adequately fit by eq 2 5 in which k B is a pH independent rat e constant for proton transfer, and (Ka)donor and (Ka)ZnH 2 O are the non interacting ionization constants of the exogenous proton donor of eq 2 2 and the zinc bound water. RH 2 O/[E] = k B /[(1 + (Ka)donor/[H + ])(1 + [H + ]/(Ka)ZnH 2 O)] ( 2 5 ) With the addition o f derivatives of imidazole and pyridine at concentrations up to 200 mM, we have observed a weak inhibition of both R1 and RH 2 O. This is probably due to binding at or in the vicinity of the zinc in the manner found for the binding of imidazole to carbonic a nhydrase I (22) or 4 MI to a mutant of H CAII (23). The binding constant Ki for this inhibition is generally greater than 100 mM, indicating weak binding at the inhibitory site. Some exogenous donors exhibited no inhibition. In each case of inhibition, a s ingle value Ki described inhibition of both R1 and RH 2 O, as determined by these equations: R1 obs = R1/(1+[B]/K i ) and RH 2 O obs = RH 2 O/(1+[B]/K i ). (2 6) An Extrel EXM 200 mass spectrometer with a membrane inlet probe (20) was used to measure the isotopic con tent of CO 2 Solutions contained 25 mM total concentration of all species of CO 2 unless otherwise indicated. All e xperiments were

PAGE 50

50 performed at 25 C and in all experiments ionic strength was held at a minimum of 0.2 M by addition of sodium sulfate. Several experiments were carried out in 98% D 2 O, and in such cases the values of pD used were uncorrected pH meter readings. The correction of a pH meter reading in 100% D 2 O [pD = (meter reading) + 0.4] is approximately offset by the change in ionization constant of acidic groups in D 2 O (pKD pKH = 0.5 0.1) for almost all acids with pK values between 3 and 10 ( 54, 57 59 60 ) Stopped f low Spectrophotometry A frequently used rapid kinetics techniques is stopped flow. Small volumes of solutions are rapidly driven from syringes into a high efficiency mixer to initiate a fast reaction. The resultant reaction volume then displaces the contents of an observation cell thus filling it with freshly mixed reagents. The volume injected is limited by the stop syringe which p achieved. The mixture entering the flow cell i s only milliseconds old. The time of this reaction volume is also known as the dead time of the stopped flow system. As the solution fi lls the stopping syringe, the plunger hits a block, causing the flow to be stopped instantaneously. Using appropriate techniques, the kinetics of the reaction can be measured in the cell (Figure 2 4) ( 61,62 ). X Ray Crystallography X ray crystallography is a method of macromolecular structure determination using the arrangement of atoms within a crystal, in which a beam of X rays strikes a crystal an d causes the beam of light to disperse into many specific directions. From the angles and intensities of these diffracted beams, a three dimensional picture of the density of electrons within the crystal can be produced. From this electron density, the mean

PAGE 51

51 positions of the atoms in the crystal can be calculated, as well as their chemical bonds, structural disorde r and other information. In an X ray diffraction measurement, a crystal is mounted on a goniometer and gradually rotat ed while being bombarded with X rays, producing a diffraction pattern of regularly spaced spots known as reflections The reflections give insight into atomic position and wave intensity. The two dimensional images taken at different rotations are convert ed into a three dimensional model of electron density using the mathematical method of Fourier transforms combined with chemical data known f or the sample. Poor resoluti on or even errors may result if the crystals are too small, or not uniform enough in their internal makeup. Crystals and X rays Crystals are regular arrays of atoms, and X rays can be considered waves of electromagnetic radiation. Atoms scatter X ray wave s, primarily through the atoms' electrons. A n X ray striking an electron produces secondary spherical waves emanating from the electron. This phenomenon is known as ela stic scattering and the electron is known as the scatterer, responsible for the diffraction, or scattering of the x rays A regular array of scatterers produces a regular array of spherical waves. These waves often cancel one another out in most directio ns through destructive interference however they add constructively in a few specific directions, determined by Bragg's law : (2 7) Here d is the spacing between diffracting planes, is the incident angle, n is any 5 ). These specific directions appear as reflectio ns on the diffraction pattern Thus, X ray diffraction results from an

PAGE 52

52 electroma gnetic wave, an x ray, colliding with a regular array of scatterers (the repeating arrangement of atoms within the crystal). X rays are used to produce the diffra typically the same order of magnitude as typical chemical bond lengths, allowing for visualization, or resolution of atomic structures. The technique of single crystal X ray crystallography has three basic steps : crystallization, data processing and refinement. The first and often most difficult step is to obtain an adequate crystal of the material under study. It is beneficial for the crystal to be large (typically larger than 0.1 mm in all dimensions), pure in composition and regular in structure, with no significant internal imperfections such as cracks or twinning A rule of thumb for optimal crystallization success is to pro duce a sample of high concentration, approximately 10 mg/mL. In the second step, the crystal is placed in an intense beam of X rays producing a regular pattern of reflections known as a diffraction pattern. As the crystal is gradually rotated, previous reflections disappear and new ones appear. The intensity of every spot is recorded at every orientation of the crystal. Multiple data sets may have to be collected, with each set covering slightly more than half a full rotation of the crystal The number of images required for the final step can also vary, depending on the symmetry of the molecule in the unit cell. In the third step, these data are combined computationally with complementary chemical information to produce a model of the arrangement of at oms within the crystal. The final, refined model of the atomic arrangement is known as the crystal structure

PAGE 53

53 The Unit Cell The theory of crystallography centers on the idea that a crystal contains an ordered arra ngements of molecules. To simplify mathem atics, the unit cell has been proposed. The unit cell is a philosophical box containing a single repeating unit of a crystal. When determining a crystal structure, the structure obtained is that of the contents of the unit cell. The idea of the unit cel l has proved to be very useful in this field in regards to data processing, calculations and data collection time. With protein molecules containing thousands of diffracting atoms, determining reflections becomes challenging. To further simplify the mat hematics, the Miller index system was developed. This proposes that the diffraction of a wave is the result of collision with a theoretical plane that goes through the unit cell. These lattice planes also go through specific atoms from where the diffract ion occurs. Each reflect ion is given a set of indices (H, K, and L ) that denote the coordinates of lattice planes that m ade that reflection Growing Crystals Protein crystals are typically grown in solution. A common approach is to lower the solubility of its component molecules gradually; if this is done too quickly, uncontrolled aggregation and precipitation occurs. Crystal growth in solution is characterized by two steps. The first step is the development of a crystallite, or small aggregate of protein molecules, known as nucleation. The following step is subsequent growth into a hopefully larger crystal. The solution conditions that favor nucleation are not always the same conditions that favor the subsequent crystal growth. The goal is to identify c onditions that favor the development of large crystals that offers improved resolution of the molecule. Two methods for growing crystals are the hanging drop and sitting drop

PAGE 54

54 methods. These methods promote nucleation through vapor diffusion. If nucleati on is favored too much, small crystallites will form in the droplet, rather than one large crystal; if favored too little, crystal formation will not occur. Several factors are known to inhibit crystallization. The growing crystals are generally held at a constant temperature and protected from shocks or vibrations that might disturb their crystallization. Impurities in the molecules or in the crystallization solutions are often detrimental to crystallization. Conformational flexibility in the molecule also tends to make crystallization thermodynamically unfavorable. Ironically, molecules that tend to self assemble into regular helices are often unwilling to assemble into crystals. Crystals can be marred by twinning, which can occur when a unit cell can pac k equally favorably in multiple orientations. The inability to crystallize a target molecule may require a modified version of the molecule; even small changes in molecular properties can lead to large differences in crystallization behavior. It is impor tant however to be cautious of the impact the modifications may have. Another concern with crystal growth is crystal packing. Crystal packing occurs when protein molecules approach positions or orientations resulting from the crystal formation, not natura l conformation. These unnatural orientations are known as crystal artifacts. Protein molecules can also endure crystal contacts, in which protein molecules interact with each other (make contact) as a result of crystal packing. When performing crystallo graphy in collaboration with other techniques, or when a certain macromolecule is studied in numerous forms through crystallography, artifacts and crystal contacts can be deduced to prevent errors in structural diagnoses.

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55 Data Processing When a crystal is exposed to an intense beam of X rays, the electrons surrounding the atoms of the proteins in the crystal scatter the X rays into a pattern of reflections that can be observed on a screen behind the crystal. The relative intensities of these spots provide the information needed to determine the arrangement of molecules within the crystal in atomic detail. The intensities of these reflections may be recorded with image plates an area detector or with a charge coupled device (CCD) image sensor. The peaks at small angles correspond to low resolution data, whereas those at high angles represent high resolution data; thus, an upper limit on the eventual resolution of the structure can be determined from the first few images. One image of reflections is insuffic ient to determine the entire structure; it represents only a small slice of the full Fourier transform. To collect all the necessary information, the crystal must be rotated step by step through 180 degrees, with an image recorded at every step. If the cr ystal has a higher symmetry, a smaller angular range such as 90 or 45 degrees may be recorded. The recorded series of two dimensional diffraction patterns, each corresponding to a different crystal orientation, is converted into a three dimensional model of the electron density; the conversion uses the mathematical technique of Fourier transforms, which is explained below. Each spot corresponds to a different type of variation in the electron density; the crystallographer must determine which variation cor responds to which spot (indexing), the relative strengths of the spots in different images (merging and scaling) and how the variations should be combined to yield the total electron density (phasing).

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56 Data processing begins with indexing the reflections. This means identifying the dimensions of the unit cell and which image peak corresponds to which position in reciprocal space. A byproduct of indexing is to determine the symmetry of the crystal, i.e., its space group. Having assigned symmetry, the data i s then integrated. This converts the hundreds of images containing the thousands of reflections into a single file, consisting of records of each reflection, and an intensity measurement for each reflection. At this state the file often also includes erro r estimates and measures of partiality, which is a parameter indicating reflections that were not fully recorded and what part of a given reflection was recorded on that image. A full data set may consist of hundreds of separate images taken at different p ositions of the crystal. The objective now is to identify which peaks appear in two or more images (merging) and to scale the relative images so that they have a consistent intensity scale. Optimizing the intensity scale is critical because the relative in tensity of the peaks is the key information from which the structure is determined. The repetitive technique of crystallographic data collection and the often high symmetry of crystalline materials cause the diffractometer to record many symmetry equivalen t reflections multiple times. This allows calculating the symmetry related R factor, a reliability index based upon how similar are the measured intensities of symmetry equivalent reflections, thus assessing the quality of the data. The recorded series of two dimensional diffraction patterns, each corresponding to a different crystal orientation, is converted into a three dimensional model of the electron density using the mathematical technique of Fourier transforms. Each spot corresponds to a different ty pe of diffracted atom, adding variation to the electron

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57 density; the crystallographer must determine which atomic variation corresponds to which spot, the relative strengths of the spots in different images and how the variations should be combined to yiel d the total electron density. Phasing The data collected from a diffraction experiment is a reciprocal space representation of the crystal lattice. The position of each reflection is governed by the size and shape of the unit cell, and the inherent symmet ry. The intensity of each reflection (spot) is recorded, and this intensity is proportional to the square of the structure factor amplitude: I = F 2 = F exp ( i hkl ) (2 8) Here I represents the intensity, and F is the structure factor amplitude. The stru cture factor is a complex number containing information relating to both the amplitude and phase ( ) of a wave. In order to obtain an interpretable electron density map, both amplitude and phase must be known. This is vital because an electron density map allows a crystallographer to build a starting model of the molecule. The phase cannot be directly recorded during a diffraction experiment, so methods of determining Initial phase estimates have been developed. A popular method for phase determination is molecular replacement. If a related structure is known, it can be used as a search model in molecular replacement to determine the orientation and position of the molecules within the unit cell. The phases obtained this way can be used to generate electr on density maps: (hkl)) (2 9) V the structure factor amplitude F hkl positions x, y, z of reflections, the phase of the

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58 w ave and the Miller indices of lattice planes h, k, l. Having obtained initial phases, an initial model can be built. This model can be used to refine the phases, leading to an improved model. Given a model of some atomic positions, these positions and the ir respective B factors (temperature factors accounting for the thermal motion of the atom) can be refined to fit the observed diffraction data, ideally yielding a better set of phases. A new model can then be fit to the new electron density map and a furt her round of refinement is carried out. This continues until the correlation between the diffraction data and the model is maximized. The agreement is measured by an R factor defined as the agreement between the crystallographic template model and the x ra y diffraction data. While Rwork measures agreement between observed data and a refined structure, a similar quality criterion is Rfree, which is calculated from a subset (~5 to 10%) of reflections that were not included in the structure refinement. Both R factors depend on the resolution of the data. As a rule of thumb, Rfree should be approximately the resolution in angstroms divided by 10; thus, a data set with 1.5 resolution should yield ~ 0.15 for the final Rfree value. Phase bias is a serious probl em in such iterative model building. Omit maps are a common technique used to check for this. It may not be possible to observe every atom of the crystallized molecule. Electron density is an average of all the molecules within the crystal, and the elect ron density for atoms existing in multiple conformations is smeared to such an extent that it is no longer detectable in the electron density map. Weakly scattering atoms such as hydrogen are routinely invisible. An example of an electron density map arou nd a refined model is shown in Figure 2 6 ( 63 65 )

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59 Figure 2 1. Diagram of steps involved in Polymerase Chain Reaction (PCR) F igure made from Pulst et al (56 )

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60 Figure 2 2. Site Directed Mutagenesis involves the use of PCR for synthesis of mutant DN A, followed by digestion of nonmutated parental DNA. Figured based off Directed Mutagenesis

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61 Figure 2 3. Carbon ic Anhydrase catalyzed oxygen 18 exchange at chemical equilibrium

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62 Figure 2 4. Basic setup of a stopped flow spectrophotometry experiment. Two driving syringes push reactants into a mixing chamber. The reaction causes an effect analyzed by the spectrometer. Figure generated from Department of Chemistry at the University of Malta Availa ble from http://staff.um.edu.mt/jgri1/teaching/ch237 (66)

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63 Figure 2 5 and 3 equals a full wavelength of waves inscribed in the scatterers (spots). Thus, constructive interference occurs, resulting in a strong reflection Figure based off of work from W.L. Bragg ( 67 )

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64 Figure 2 6 Electron Density Map of V143A. PDB ID: 3U45 (unpublished) Figure was made using PyMol

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65 CHAPTER 3 CAII AND CYANATE BIN DING Anion Binding and Metal S ubstitution in HCAII The binding of anions in the active site of CA has been well studied. H CA II is inhibited by a wide array of molecule s, many of them anionic in nature ( 72,74 ). The affinities of anions for the active site of H CAII have been attributed to their interaction with the central metal atom. This has been shown by studies of C AII with the substituted metals, for example CA whe re zinc was replaced with cobalt, manganese, nickel, and copper, all in a + 2 ionization state (3). The main reason for finding a metal to substitute for zinc is to find a metal in which ligand binding can be studied by absorption spectroscopy and NMR. Zi nc has a very weak nuclear magnetic moment and is diamagnetic (creates magnetic fields and repelled by externally applied magnetic fields)( 68 ) which would hinder many spectroscopic experiments. There are few metals that can replace zinc in CA and yield an active enzyme. Cobalt is the only metal that provides the enzyme with activity closest to the level of wild type ( 3 ). Inhibitor Coordination in A ctive CAs Structurally it has been shown that m any CA inhibitors (CAIs) bind near the amine group of T199 be tween the metal and hydrophobic region (substrate binding pocket) of the active site ( 6 9 ). The hydrophobic region is essential to the binding of substrate CO 2 ( 40,41,70 ) and as a result, this site is highly conserved throughout various isozymes and is res istant to changes in activity aside from mutations resulting in the introduction of a charged or bulky residue ( 40 ). Similarly, it is also proposed that the hydrophobic region is responsible for the reduction of binding and inhibition strength of anions d ue to the lack of h ydrogen bond interaction (71 ). In regards to coordination with

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66 wild type H CAII, various binding modes have been observed: tetrahedral coordination, distorted tetrahedral coordination, penta coordination, and competitive binding (inhibit ion without binding to the metal, but near the vicinity of substrate binding) (3 ). Tetrahedral c oordination Tetrahedral coordination is seen in CAII involving the three stab ilizing histidines and a zinc bound solvent (Chapter 1). Anions known for this co ordination are sulfonamides, a well known class of CAIs Single atom anions, including iodide and bromide, illustrated in Figure 3 1, show similar coordination. Another cl ass is sulfites (3 ) as well as substrate bicarbonate ( 4 3 ). The protonated oxygen ( the hydroxyl group) of sulfites and acetate (Figure 3 2) bind in the same position as the amine group in sulfonamides: hydrogen bonded to T199. Studies show that azide binds to the zinc tetrahedrally in H CAII ( 72 ) but with a distorted conformation (Figu re 3 3). This leads to the other binding mode: distorted tetra coordination. There are cases in which anions are unable to donate hydrogen bonds to T199. This means that they are only able to bind to the metal, leading to a distorted position. T hey a re also able to displace the deep water molecule and occupy the hydrophobic cavity. The anions that promote this type of binding are likely high in polarity ( 3) Penta coordination This coordination is seen occasionally with zinc and frequently with C o C As In this binding mode, the anion typically contains a carboxyl group that can accept a hydrogen bond from the amine group of T199, while the carboxyl oxygen of T199 binds closely to the metal bound water. The position of the zinc bound solvent is move d almost 1 from its natural position, resulting in both the zinc bound water and inhibitor

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67 H C A II in complex wi th formate forms a penta coordinated complex (Figure 3 4) ( 3 2 3 74 ). Uncoordinated binding This is the binding mode for anions that have a very weak affinity for the zinc atom, or any coordinated metal in the active site. They still bind in the hydrophobic region of the active site but do not perturb the tetra co ordination or displace the zinc bound water. However, they typically can form hydrogen bonds with T199. Examples of these in hibitors include nitrate ( 74 ), cyanide, and cyanate (7 5 ), an anion which will be discussed in further detail. NMR Experiments Pro mote Controversy with Cyanate Binding NMR experiments on cobalt substituted and wild type CA have been used to study binding affinity of anions ( 76,77 ) In studies with bovine CAII, NMR spectra suggest that cyanate and cyanide bind to the zinc atom formi ng a tetrahedral complex, thereby replacing the water molecule. Similar attempts with cyanide and cyanate were pe rformed when crystal structures showed that these anions do not bi nd directly to the zinc These spectra indicate tetrahedral adduct formati on of cyanate and cyanide, even in isotopic and natural zinc CA ( 3 ). Structural Data Promotes That Cyanate Is a Zinc bound Ligand The work presented here outlines the behavior of cyanate in the active site of ( HCAII). Results presented focus on HC AII crys tal structures wtHCAII and V207I HCAII each enzyme with cyanate bound in the active site. Affinity measurements for these two mutants as well as V143I, a mutation known to impact binding in the hydrophobic patch of the active site, are presented. The r esults show that cyanate can in fact act as an inhibitor binding directly to the zinc in place of zinc bound solvent for

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68 both structures. Affinity constant measurements show similar binding affinity for w tHCAII and V207I HCAII, and a decrease for V143I HC AII This data provides powerful insight into cyanate as an inhibitor for HCAII. Comparisons with other structures containing anions in various conformations are made to further illustrate the position and coordination of cyanate as well draw possible ex planations to the inconsistency of various binding studies, and the possibility of mu ltiple coordination states. These data will hopefully shed light on the binding behavior of cyanate, and possibly similar anions. Materials and Methods Expression and Pur ification of Mutants. Please s ee Chapter 2 sections Site Directed Mutagenesis, Protein Expression, and Affinity Chromatography for more information Crystallization. Crystals of the HCAII mutants were obtained using the hanging drop vapor diffusion me thod. The drops were prepared by mixing 5 L of protein at a concentration of 10 mg/mL in 50mM Tris HCl (pH 8.0) with 5 L of the precipitant solution containing 50 mM Tris HCl (pH 8) and 1.4 M sodium citrate against 1 mL of the precipitant solution. Th e drops were allowed to equilibrate at room temperature. Crystals were stored in polystyrene containers and allowed to sit undisturbed. Useful crystals were observed three days after setup. Data Collection The X ray diffraction data sets for the HCAII mutant crystals were obtained using the oscillation method in house at 120C, using a Rigaku RU H3R Cu rotating anode operating at 50 kV and 100 mA. Diffraction was detected using an R AXIS IV++ image

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69 plate system. The crystal detector distance was s et to 80 mm. The crystals were soaked in a 0.01M solution of Sodium Cyanate, followed by cryoprotectant solution of glycerol (30%) and 1M Tris HCl (70%) and mounted on cryo loops. The oscillation steps were 1 with a 5 min exposure time. Structure Soluti on and Model Refinement The H CAII variant structures were solved using the program phenix.refine in PHENIX. Preceding refinement, random test sets of ~10% were flagged for Rfree calculations. The method of structure determination was molecular substitut ion (phaser) with w t HCAII (PDB ID: 2ILI). The w t structure, stripped of the zinc, solvent and amino acid Val 207 replaced with Ala, was used as the initial model for phase calculations in phenix.refine. The cyanate ligand was designed usin g ProDrug, and c if files needed for refinement were made using PHENIX. Following 3 cycles in PHENIX, the refined structure was visually inspected using the molecular imaging system Coot for display of model and electron density. The zinc, the amino acid substitution at position 207, as well as improperly positioned side chains, were manually placed in their respectively density. This refined model was then submitted for subsequent rounds of refinement and solvent placement. During the final stages of refinement in conjun ction with COOT, the models were viewed and solvent with little or no 2F0 FC density were deleted, until the Rwork and Rfree values had converged (Table 3 1) Enzyme Titration : Inhibitor Affinity For strong binding anions like cyanate, when K i >> [E], it obeys the equation of A = Ao /(1+[I]/ K i ) where A is activity at various Inhibitor concentrations and Ao is the initial activity without inhibitor. Inhibitor affinity was measured by enzyme titration using an Extrel EXM 200 mass spectrometer. The uncata lyzed and carbonic anhydrase

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70 catalyzed exchanges of 18 O between CO 2 and water at chemical equilibrium (a flat line of current for all CO 2 species) were measured at room temperature in the absence of buffer at a total substrate concentration of 25 mM. HCAI I was added to the solution. After 2 minutes, cyanate was added to the reaction until the reaction regained equilibrium indicating the inhibition of HCAII. Calculations of affinity from experimental data were performed using Enzfitter (Biosoft). Results Crystal Structures The binding site of cyanate in three variants of H CAII was determined by X ray crystallography refined to below 1.8 resolution with final R factors of 17.2 and 13.9 (See Table 3 1). Each structure clearly shows cyanate bound directly t o the zinc with oxygen interacting with the metal ion. The cyanate ion, labeled OCN faces into the hydrophobic pocket, approximately 2 from the zinc atom. The zinc bound solvent is not present in any of the crystal structures indicating that the cy anate has replaced it. There appears to be no changes in side chain conformation caused by inhibitor binding or mutations. Low r.m.s.d. values for bond lengths and angles shows that there are negligible perturbations in the structure and the bond lengths and angles are highly accurate ( Table 3 1). Each structure shows cyanate in its natural linear conformation. Both cyanate structures show that the amine group of the anion is in hydrogen bonding distance (2.6 to 3.5 ) with the hydroxyl group of T199, s imilar to other tetrahedral coordinated ani ons (Figure 3 5 and Figure 3 6). The overlay of all variants shows that the cyanate is bound in the same proximity of the zinc atom for each structure (Figure 3 7). The binding of cyanate is further illustrated when compared to anions hydrogen

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71 sulfide, azide and bisulfite that also form tetrahedral complexes with three histidines stabil izing the zinc atom (Figure 3 8). Inhibitor Affinity The tightness of binding of cyanate to each variant of HCAII was examined by 18 O exchange between CO 2 and water measured by mass spectrometry. The inhibition in 18 O exchange rates are shown in Figure 3, with resulting values of K i given in Table 2. The binding of cyanate to V143I H CAII is lower than for wt and V207I HCAII (Tabl e 3 2). These values are compared to previously determined inhibition constants for w tH CAII as well as cobalt substituted CAII. While the cobalt CAII is the lowest at 7 M, all of the variants listed have values in the M range possibly making their diff erences insignificant as they are all very strong inhibitors. Discussion The work with cyanate bound structures of HCAII was done initially as a method to study the possible change in substrate conformation dependent upon mutations of specific residues in the CO 2 binding pocket. This was a strategy to screen for mutants (V143I and V121I) that had interesting results that would be further studied with CO 2 entrapment. When the structure of each mutant was solved, it appeared that cyanate did not bind near CO 2 but rather it bound directly to the zinc (Figure 3 9). The cyanate is oriented approximately 2 away from the zinc similar to the natural hydroxide. Interesting is the fact that these mutations were developed in the theory that they would impact su bstrate binding and the most significant deviation from wild type in regards to affinity for cyanate came from V143I, showing binding affinity can be altered due to mutations.

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72 Structurally, it appears that cyanate c an bind to zinc forming a tetra hedral co ordination complex. Superimposition of CA inhibitors with similar behaviors verifies this claim (Figure 3 8). This anion appears to be in hydrogen bond distance with T199, a common factor in regular tetrahedral coordination. However, if the oxygen is bo und to the zinc, the nitrogen, with a full shell of valence electrons, would make an unlikely hydrogen bond acceptor. The coordination of cyanate appears to be a distorted tetrahedral. The affinities of our CA variants for cyanate correlate closely with other data documenting Ki of similar metal oxides with various other carbonic anhydrases (8 0 ), as well as with previous work that shows I143 having an impact on liga nd binding in the active site(40 ). Inhibitor constants for cyanate were determined by Lin dskog et al. fo r cobalt substituted CA (79 ). The values of the inhibition constants for cyanate match closely to the ones obtained by Supuran et al. (78 ) as well as measured for w t V143I, and V207I H CAII variants from this current study (Table 3 2). The affinity of cyanate for cobalt CAII was measured to be about 7 M, while the previously mentioned variants were measured to be around 36 M. This is a promising finding, as low affinity for the central metal was a proposed reason for uncoordinated bindin g in the active site, the mode of binding that was proclaimed for cyanate in regards to zinc CAII ( 3 ). With cobalt and zinc CAs having similar values for affinity constants, it is likely that their binding tendencies are similar as well. Since it has bee n sho wn through NMR that cyanate bind s cobalt, it is possible that it also binds zinc as well. The question that still remains is the binding position of cyanate. Is the oxygen or nitrogen coordinated to the zinc? Structures of many CA Is indicate that ni trogen indeed

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73 has the ability to bind to zinc. It is postulated that cyanate binds to the zinc in H CAII with the oxygen directly coordinated to the metal. When comparing the ionization states of cyanate and hydroxide, there are similarities, primarily th e number of electrons for the oxygen atom. Both atoms have 7 electrons, adding the negative charge to the molecule. The oxygen in hydroxide binds directly to the metal, and it is proposed that the same occurs for cyanate. While the data indicates an alte rnate binding mode for cyanate in CAII, it does not necessarily disprove previous work. It is possible that cyanate can have multiple coordination possibilities. Some structures of CA with bicarbonate indicate that it can form a penta coordination comple x with water still attached (3 74 ). However it has also been shown that that bicarbonate binds in place of water as well ( 43 ). The goal of this study was to use crystallography to see if cyanate can bind directly to zinc, providing more structural eviden ce to the nature of this anion. Cyanate has the ability to bind directly to the zinc atom, replacing the water, however it is not a disproval of previous works, as our experiments were not set up to match previous crystallographic approaches. Based on da ta presented here, it appears that binding of cyanate in the active site of HCAII is that of a solvent displacing ligand rather than a weak substrate bound in the mode of CO 2 The kinetic data on these mutants designed to disrupt substrate binding shows c hange in affinity of cyanate at the metal, however the structures of cyanate bound to HCAII all show the same conformat ion, bound to the zinc (Figure 3 7 ). The interaction may be similar to that between zinc and hydroxide/water: a dipolar bond. Like hydr oxide, the oxygen molecule in cyanate has two unshared electrons. This anion

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74 also forms a tetrahedral complex, similar to that of the zinc bound water, bicarbonate, and many sulfonamides. This follows our observation that cyanate is an inhibitor of HCA II by binding to the metal, preventing the formation of zinc bound hydroxide. This is an unexpected result, as the belief was cyanate would bind competitively, due to previous works claiming cyanate to be a weak substrate for HCAII binding in a similar po sition as natural substrate ( 75 ) It is therefore our belief that cyanate, while similar in electron configuration and conformation to CO 2 does not bind in an uncoordinated orientation in HCAII, rather it binds directly to the zinc, displacing the zinc b ound solvent, inhibiting the enzyme.

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75 Table 3 1. Data and refinement statistics for cyanate bound to variants of HCAII Parameter Wild Type V207I PDB code 4E5Q n/a Space group P2 1 P2 1 Unit cell parameters a,b,c (), ( ) 42.2,41.0, 71.5; 104.5, 42.4,41.5,7 2.5; 104.4 Resolution () 20 1.7 (1.76 1.70) 35 1.5 (1.55 1.5) R sym a (%) 8.8 (5.5) 4.4 (2.5) 16.5 (4.0) 28.1 (5.2) Completeness (%) 91.1 (89.2) 92.2 (87.9) Average Redundancy 6.8 (7.0) 3.8 (3.7) Number of unique r eflections 23 839 38 496 R c ryst b /R free c (%) 17.2/20.0 13.9 / 17.1 No. atoms Protein 2 090 2 073 Water 287 320 B factors ( 2 ) main/side, solvent 13.2/17.4 24.8 13.8/17.4 25.4 R.M.S.D. 0.006/1.092 0.010/1.3 Ramachandran plot (%) Most f avored allo wed, outlier 88.4, 11.6, 0.0 88.0, 12.0, 0.0 aR sym manner as bRcryst, except that it uses 5% of the reflection data omitted from refinement. *Values in parenthesis represent highest resolution bin.

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76 Table 3 2. Inhibition constants for cyanate bound in HCAII variants a Variant of HCAII Affinity Coefficient (Ki) (M) Cobalt CAII b 7 Wild type c 20 Wild type 37 + 3 V143I 28 + 1 V207I 42 + 3 a The constants were meas ured at 25 C in solutions containing 25 mM CO 2 b This data comes from Lindskog et al. ( 79 ) c This data comes from Lindahl et al. ( 75 )

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77 Figure 3 1. H CAII in complex with bromide. Many anionic inhibitors, like bromide form a tetrahedral coordination comp lex with the zinc atom, like hydroxide/water (PDB ID: 1RAZ). This model was made using PyMol (72)

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78 Figure 3 2. H CAII in complex with sulfite (SO3, PDB ID: 2CBD) and acetate (ACT, PDB ID: 1XEG). Anionic CA inhibitors with tetrahedral coordination are stabilized by hydrogen bonding with T199. This model was made using PyMol (74,81)

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79 Figure 3 3 H CAII in complex with azide. The binding mode for azide (N3) is distorted tetrahedral since is unable to donate a hydrogen bond to the hydroxyl group of T19 9 (PDB ID: 1RAY). This model was made using PyMol (72)

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80 Figure 3 4. H CAII in complex with formate. The binding of formate (FMT) is not strong enough to impede the water/hydroxide bound to zinc. As a result formate and water bind to form a penta coordi nation complex. Dotted red lines indicate bonds in the range of hydrogen bond distance (2.6 to 3.5 ). PDB ID: 2CBC. This model was made using PyMol (74)

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81 Figure 3 5. HCAII in complex with cyanate. In comparison with other tetra coordination complexe s, cyanate (OCN ) in CAII binds directly to the zinc, and is in hydrogen bond d istance with T199. PDB ID: 4E5Q This model was made using PyMol

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82 Figure 3 6. V207I HCAII in complex with cyanate. This model was made using PyMol

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83 Figure 3 7. Overlay o f wild type (yellow) and V207I (white) HCAII in comple x with cyanate. R.m.s.d. = 0.23 This model was made using PyMol

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84 Figure 3 8. Overlay of CAII/anion complexes with acetate (gray PDB ID: 1XEG ) azide (yellow PDB ID 1RAY ) sulfi te (pink PDB ID: 2C BD ) and cyanate (blue PDB ID: 4E5Q ) R.m.s.d. = 0.28 to 0.55 This model was made using PyMol

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85 Figure 3 9. H CAII with cyanate and carbon dioxide. The positions of inhibitor cyanate (green PDB ID 4E5Q ) and substrate carbon dioxide (blue) from Domsic et al (PDB ID: 3D92). This model was made using PyMol

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86 CHAPTER 4 CO 2 BINDING AND CATALYTI C EFFICIENCY This chapter focuses on binding of carbon dioxide in the active site of H CA II. As previously mentioned, the binding pocket of HCAII is composed of hydrop hobic residues: three valines (Val121, 143 and 207), Ile198, Trp209, and the amide group of Thr199. This is the region in which CO 2 binds and the first s tage of catalysis takes place ( 9, 38 ). This chapter places emphasis on structural and kinetic studies of the binding pocket residues of HCAII. The Hydrophobic Pocket Studies on amino acid substitutions of hydrophobic pocket residues in the active site of HCAII have presented the hypothesis that increasing hydrophobicity decreases catalytic efficiency and decreasing hydrophobicity promotes no significant effect. However, this may not be the case for all residues in the binding pocket of HCAII. Much work has been done categorizing these hydrophobic residues in relation to their effect on CO 2 binding and co nversion to bicarbonate. This is likely a result of a well accepted notion that the two stages of the reaction catalyzed by HCAII are separate and do not affect each other. As a result, much work focused on structural modifications of the hydrophobic reg ion is correlated with changes in catalytic efficiency (similar tendencies occur for the hydrophilic region and proton transfer). This notion however is not without its validation, as our data has shown that mutations in the hydrophobic pocket deliver no significant changes in proton transfer. However these changes, albeit minor illustrate that the reaction rates have the ability to be altered when the hydro phobic pocket is manipulated ( 40 ). It has been shown that mutations in the binding pocket affect s ubstrate conversion, while mutations altering the water network impact proton transfer

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87 ( 8 2, ). However, the inverse is not thoroughly considered: can mutations in the CO 2 binding pocket significantly affect proton transfer? Can mutations in the hydrophili c region impact the dynamics of catalytic efficiency? Although the binding of CO 2 in the active site of H CAII has been examined by X ray crystallography ( 38 4 3 ), the relevance to the catalysis of its position in the enzyme substrate complex is not well un derstood. Little is known about the relationship between catalytic efficiency and substrate orientation, a relationship difficult to elucidate experimentally. The work presented here focuses on catalysis by variants of H CAII and examines using X ray crysta llography the binding of CO 2 and bicarbonate in the active site of V143I H CAII The presence of CO 2 at high pressure lowers pH and promotes zinc bound water which is not reactive with CO 2 The variant V143I H CAII had catalysis decreased about 17 fold compa red with wild type. This is a change unseen by other binding pocket mutants. The larger side chain of Ile143 in V143I decreases the size of the active site cavity and forces bound CO 2 closer to the zinc by 0.3 This implies a perturbed, crowded transiti on state and an impeded formation of developing charge on product bicarbonate by increased hydrophobicity of Ile143. There is no significant effect on the structure of ordered water in the active site cavity in V143I H CAII and there are only small effects on rate constants for proton transfer. These structural and kinetic studies elucidate the significance of Val143 and the CO 2 binding site in maximizing catalysis. Materials and Methods Expression and Purification of Mutants. Please see Chapter 2 Section on Protein Expression Site Directed Mutagenesis and Affinity Chromatography.

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88 Crystallization. Crystals of H CAII variants were obtained using the hangin g drop vapor diffusion method (8 3). The drops were prepared by mixing 5 L of protein (~10 mg/mL con centration) in 50mM Tris HCl (pH 8.0) with 5 L of the precipitant solution containing 50 mM Tris HCl (pHs 8, 8.5, and 9) and 1.3 to 1.4 M sodium citrate against 1 mL of the precipitant solution. The drops were allowed to equilibrate at room temperature. T he crystals trays were then stored and allowed to sit undisturbed for one week (8 3). A crystal of each H CAII variant was cryoprotected by quick immersion into 30% glycerol precipitant solution and flash cooled by exposure to a gaseous stream of nitrogen at 100K. CO 2 Binding. CO 2 entrapment experiment for a V143I H CAII crystal was achieved by rapid cryo cooling at 15 atm pressure of CO 2 A crystal was soaked in a cryo solution containing 20% glycerol in precipitant solution, then coated with mineral oil and loaded into a high pressure tube. In the pressure tube, the crystal was pressurized with CO 2 gas at 15 atm at room temperature and cryo cooled by being plunged into liquid nitrogen ( 84 ). Data Collection. X ray diffraction data for the H CAII variants wer e obtained using an in house R AXIS IV++ image plate system with Osmic Varimax HR optics and a Rigaku RU H3R Cu rotating anode operating at 50kV and 22 mA. The detector crystal distance was set to 76 mm. The oscillation steps were 1 with a 5 to 7 min expo sure per image. Indexing, integration, and scaling were performed using HKL2000 (8 5). All three data were collected had an average completeness of < 90% and linear R factors of > 5.0% (Table 4 1).

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89 Diffraction data for the CO 2 bound V143I H CAII crystal was collected at Cornell High Energy Synchrotron Source (CHESS) using a wavelength of 0.9772 Data were collected in 1 oscillation steps with a 2 s on an ADSC Quantum 210 CCD detector (Area Detector Systems Corp.), with a crystal to detector distance of 65 mm. The data collected had average completeness of 92.3% and lin ear R factors of 8.2% (Table 4 1) Structure Solution and Model Refinement. All three in house H CAII variants structures were solved using the programs phaser and phenix.refine in PHENIX ( 86 ) Preceding refinement, random test sets of ~5% were flagged for Rfree calculations. The method of structure determination was molecular substitution (phaser) with wild type H CAII (PDB ID: 2ILI) The wild type structure, stripped of the zinc, solvent and a mino acid Val 143 replaced with Ala, was used as the initial model for phase calculations in phenix.refine. Following 3 cycles in PHENIX, the refined structure was visually inspected using the molecular imaging system Coot for display o f model and electron density The zinc, the amino acid substitution at position 143, as well as improperly positioned side chains, were manually placed in their respectively density. This refined model was then submitted for subsequent rounds of refinement and solvent placeme nt. During the final stages of refinement in conjunction with COOT, the models were viewed and solvent with little or no significant 2F0 FC density were deleted, until the Rwork and Rfree values had converged (Table 4 1). The CO 2 V143I H CAII structure was similarly refined, but with the addition of dual occupancy refinement of the both bound CO 2 and bicarbonate (Table 4 1). 18 O Exchange Mass Spectrometry See Chapter 2 Mass Spectrometry with HCAII.

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90 Stopped Flow Spectrophotometry Initial rates of CO 2 hydrati on were determined by measuring the change in absorbance of a pH indicator on an Applied Photophysics SX.18MV stopped flow spectrophotometer using the method of Khalifah (61 ). The pK a value and wavelength for the pH indicator buffer pair used were m cresol purple (pK a TAPS. Final buffer concentrations were 25 mM and the pH was 8.3. CO 2 solutions were prepared by bubbling CO 2 into water at room temperature (25 C) at varying CO 2 concentrations. The mean in itial rates were measured from 5 to 8 reaction traces comprising the initial 10% of the reaction. Uncatalyzed rates were determined similarly and subtracted from the total observed rates. Determinations of the constants k cat and k cat /Km were carried out using Enzfitter (Biosoft). Results Catalysis Catalysis by H CAII variants of the exchange of 18 O between CO 2 and water was measured by membrane inlet mass spectrometry. Compared with wild type H CAII each of the variants had decreased catalytic efficiency as measured by k cat exch /K eff CO 2 (Table 4 2). Both mutants V143I and V143L showed sizable decreases in maximal, pH independent value of k cat exch /K eff CO 2 by as much as 17 fold. The variant V143A decreased this efficiency about two fold compared with wild t ype. Changes in the rate constant for proton transfer k B were two fold at most (Table 4 2). The values of the pK a of the zinc bound water molecule obtained from the pH profiles of k cat exch /K eff CO 2 (Figure 2) and RH 2 O/[E] (Supporting Information, Figure 4 1) were not significantly altered compared with wild type (Table 4 2). However, the values of the pK a of the imidazole side chain of His 64 obtained from pH profiles of RH 2 O/[E] were decreased slightly for

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91 the mutants (Table 4 2), an effect seen previou sly in variants of H CAII in which the orientation of the si de chain of His64 was inward (87 ). The maximal values of the steady state catalytic constants for hydration of CO 2 were also measured for these variants by stopped flow spectrophotometry at pH 8.3 These data show the same trend as the 18 O exchange data, with k cat /K m for V143I and V143L decreased up to 20 fold compared with wild type, while the decrease for V143A is near three fold (Table 4 3). The values of k cat /K m determined by stopped flow shoul d in principle be the same as k cat exch /K eff CO 2 determined in the 18 O exchange experiments. Comparison of these values in Tables 4 2 and 4 3 show values which are similar. Crystal Structures. Using X ray crystallography, structures of H CAII variants V143A, V143L and V143I, were solved at 1.7, 1.6, and 1.55 resolution, respectively. The structures were refined with final R factors of < 18% (Table 4 1). The side chains of each of the substituted amino acids at position 143 faced into the active site, in a m anner similar to w tHCAII (Figure 4 3). When comparing the overall structures of th e three variants with wt H CAII (PDB: 2ILI) (10), only very minor alterations were noticed based on the structural overlay, which showed an average r m s d of 0.19 between C four superimposed models (Figure 4 4). For the variant V143A H CAII the size of the CO 2 binding pocket was increased, and two additional ordered solvent molecules (labeled WA and WB in Figure 4 3A) were observed. These water molecules are no t observed in wild type or in the other two variants (Figure 4 3B,C) In addition to these extra solvent molecules in V143A H CAII and most likely as a consequence of their 3) appears 2.7 away from the oxygen atom of the zinc bound solvent compared to 2.4 for wt H CAII In

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92 the variant V143L, the deep water appears to have dual occupancy and is shifted 1.0 from the zinc (shown as two red spheres labeled DW in Figure 4 3 C ). Substrate and P roduct Binding in V143I H CAII Following the procedures of Domsic et al, ( 38 ) for the entrapment of CO 2 in w t H CAII we examined substrate binding in V143I H CAII at a resolution of 1.3 With an r m s d V143I variant, superimposition shows good structural agreement. Interestingly, both the substrate CO 2 and product bicarbonate were observed in the active site (Figure 4 5), this is in contrast to the wild type structure where only CO 2 was observed under th ese conditions. Hence, careful refinement was performed to determine occupancy of the CO 2 (0.33), the zinc bound solvent (0.33), and bicarbonate (0.67). Similar to the wild type structure by Domsic et al, His64 has a dual in ward and outward conformation (3 8 ), an unpredictable state when alterations to HCAII have been introduced. Figure 4 5A and 4 5B show the calculated omit Fo Fc electron density map when the CO 2 or bicarbonate were not incorporated into the respective models. This clearly demonstrates the occupancy of both substrate and product. The CO 2 molecule bound in V143I H CAII is oriented in a side on conformation similar to that observed by Domsic et al ( 3 8 ) (Figure 4 6A ,C ). The CO 2 molecule shows similar spatia l orientations in both wt and V143I H C AII but with the CO 2 slightly tilted ~10 in the direction of O 2 The central carbon atom of CO 2 is noticeably closer to the oxygen atom of the zinc bound water, now 2.5 away compared to 2.8 for w tHCAII (Figure 4 7A, Table 4 4). The bicarbonate is pos itioned with O1 and O3 proximal to the CO 2 1.2 away from corresponding oxygen atoms of bound CO 2 (Figure 4 6B). The other oxygen of the

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93 bicarbonate, O2, overlaps with the zinc bound solvent observed in the CO 2 bound form, and coordinates with the zinc, 6B, Table 4 4). the CO 2 and the zinc bound water. The bicarbonate position observed in V143I H CAII overlaps significantly when superimpos ed with the previously reported coordinates of bicarbonate bound in H CAII T200H (PDB ID: 1BIC)(21)(Table 4 4). The r m s d of all CAII structures with bicarbonate is 0.33 (Figure 4 7B). The V143I mutant has an 18o rotati on of bicarbonate toward the O2 atom compared with T200H H CAII There is also a 0.4 separation in carbon atoms between the two superimposed bicarbonate molecules in the T200H and V143I variants. Discussion With catalysis nearly diffusion controlled (k c at /Km at 10 8 M 1 s 1 (94 )) and the binding of CO 2 very weak (K d near 100 mM, ( 70 )), H CAII may not have a single well defined enzyme substrate complex but an array of CO 2 binding modes that leads to catalysis. An aim of this current report wa s to elucidate the role of the active site configuration in H CAII by perturbing the catalytic binding site of CO 2 Previous work demonstrated that specific replacements of amino acids in the hydrophobic binding pocket of CO 2 are associated with changes in catalytic rates of H CAII ( 40,70 ). Fierke et al. showed decreases in k cat /K m for hydration of CO 2 associated with the replacement of Val143 at the CO 2 binding site of H CAII by each of nine amino acids ( 40 ). The variants of H CAII at residue 143 examined in this report m ake alterations in steric volume of the active site cavity while avoiding excessive structural perturbations. The replacement of Val143 in wild type (side chain volume 140 3 ) by Ile (167 3 ) and Leu (167 3 ) demonstrate the effects of decreased steric vol ume of the cavity and

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94 increased hydrophobicity. Val143 was also replaced by Ala (89 3 ) increasing the volume of the cavity. An overlay of crystal structures of all variants examined in this work showed a superposition of side chain conformations at positi on 143 with strong side chain overlap in the residues of the active site cavity, indicating that the unaltered residues in the hydrophobic pocket were not significantly affected by the changes in the volume of the active site (Figure 4 4). Since these repl acements do not impinge on the proposed proton transfer pathway (1), there were rather minor effects on rate constants k B and k cat that contain contributions of proton transfer between the active site and solven t (Tables 4 2, 4 3)(1, 6 ). The catalytic ef ficiency, measured by k cat exch /K eff CO 2 (Table 4 2) and k cat /K m (Table 4 3), was decreased as much as 20 fold for the replacements of Val143 by side chains of larger size (Leu, Ile), and to a much lesser extent with the smaller size (Ala). These data are in rather close agreement with the stopped f low results of Fierke et al. ( 40 ) It is interesting that even increasing the volume of the active site cavity in V143A H CAII caused a decrease in catalytic activity (Tables 4 2, 4 3). In the active site, the hydro gen bond between the zinc bound hydroxide and side chain hydroxyl of Thr199 orients the lone pair electrons of the zinc bound hydroxyl towards the carbon of CO 2 promoting nucleophilic attack and avoiding an entropic penalty by immobilizing the rotation of the zinc bound hydroxide about the Zn O axis ( 42,44,45 ). In the crystal structure of CO 2 bound in V143I H CAII the solvent ligand of the zinc must be a water molecule or reaction would occur. In this structure, the larger side chain volume of Ile143 compar ed with Val has forced the carbon atom of bound CO 2 closer to the zinc bound solvent oxygen by a distance of 0.3 (Figure 4 7A, Table

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95 4 4), and this is associated with a 13 to 17 fold decrease in k cat /K m and k cat ex ch /K eff CO 2 for hydration when V143I is c ompared with wild type (Tables 4 2, 4 3). This is most likely due to a stereoelectronic effect, one that has sterically crowded the transition state due to the increased volume of Ile143; this could be accompanied by the effects of increased hydrophobicity of Ile143 compared with Val143 (estimated at 0.5 kcal/mo l based on side chain burial ( 92 )). The overall effect on V143I H CAII is to increase by about 1.7 kcal/mol an energy barrier for catalysis that in wild type is near 10 kcal/mol. This effect is also t he case for V143L which shows similar catalytic rates as V143I H CAII (Tables 4 2, 4 3). These explanations must be viewed in the context of the decrease, although smaller in magnitude, of catalytic activity when the active site cavity is increased in volu me in the variant V143A H CAII Presumably here the position of bound CO 2 is more distant from the zinc bound solvent than in wild type, reducing the interaction between the unpaired electrons of the oxygen atom of the zinc hydroxide and CO 2 Work on the bi nding of CO 2 to V143A H CAII is in progress. In the crystal structure of V143I H CAII we observed the superimposed occupancy of bicarbonate as well as CO 2 (Figure 4 5 and 4 6). There are significant changes in the interatomic distances for the three oxygens of bicarbonate when comparing binding in V143I H CAII (Table 4 4) and other reports of bound bicarbonate. It is possible that the shifts are due to the larger side chain of isoleucine in V143I H CAII ; however, these changes may be indicative of the influence of the experiment. Previous structures of HCAII variants with bicarbonate bound diffused bicarbonate into crystals ( 11 1 ) while our data show the appearance of bicarbonate after the reaction of CO 2 and at the active

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96 site. Of course, the various replacemen ts in the variants of Table 4 each could promote a change in binding that would explain the observed differences in position. Nevertheless, the bicarbonate molecules in each variant bind directly to the zinc, correlating with the catalytic mechanism of dir ect nucleophilic attack of zinc bound hydroxide on CO 2 Using conditions very similar to those reported here, Domsic et al. (38 ) did not observe bicarbonate in the crystal structure of wild type H CAII However, Sjoblom et al. ( 43 ) using repeated X ray expo sure did identify bicarbonate bound in H CAII and suggested that bicarbonate formation was a result of enzyme activation due to radiation induced events. This implies that increasing X ray dose led to enzyme activation perhaps producing active hydroxyl radi cals, which would allow for reaction with CO 2 leading to the formation of bicarbonate, a s discussed by Sjoblom et al. ( 43 ). Radiation damage is a common issue known to impact macromolecular structures under crystallographic conditions, so this behavior bet ween electrons and water molecules is not unlikely ( 88 91 ). The absorbed dose at which Sjoblom et al. observed bicarbonate was approximately 6 x 106 Gy, and the occupancies were 65% CO 2 and 35% bicarbonate. In the experiment presented here, the data collec tion for V143I H CAII maintained a constant radiation dose (estimated radiation dose absorbed at the x ray beam line used (0.97 wavelength) was 106 to 107 Gy.), and only one set of data was collected for substrate binding analysis. The occupancy levels o f substrate and product described by Sjoblom et al. are reversed in this study which found 67% bicarbonate and 33% CO 2 at the active site. The higher occupancy of bicarbonate in this study could indicate a change in the

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97 product dissociation rate for V143I H CAII A decrease in the rate of bicarbonate dissociation would explain the higher occupancy of bicarbonate as well as the decrease in k cat /K m compared with wild type. Work reported here demonstrates a role for Val143 in the hydrophobic wall providing a productive binding site for CO 2 in H CAII Replacement of Val143 with Ile resulted in a decrease in the volume of the active site cavity and a resulting shift in which the position of the carbon in bound CO 2 was 0.3 closer to the zinc bound solvent. This was accompanied by a 17 fold drop in catalysis compared with wild type. These results are consistent with the binding of CO 2 to the hydrophobic wall including Val143 as productive in catalysis and with the idea that the enzyme has evolved at residue 143 to its most efficient state (19,40) These data help to develop a possible structure function relationship between the position of substrate in the enzyme substrate complex and the catalytic efficiency. Observing specific changes in the binding of substrate related to catalytic activity provides insight into the catalysis and may provide pathways to variants of carbonic anhydrase with specifically engineered catalytic activity for industrial and environmental uses.

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98 Table 4 1 Data processing and refinement s tatistics for the structures of the H CAII variants Parameter V143A V143I V143L V143I (CO 2 bound) PDB code 3U3A 3U45 3U47 3U7C Space group P2 1 P2 1 P2 1 P2 1 Cell dimensions a,b,c ( ); ) 42.3, 4 1.6,72.3 ;1 0 4.4 42.2, 4 1.3,72.1; 1 04.2 42.3,41.6,7 2.3 ; 104.5 42.3 ,41.5,7 2.1; 104.2 Resolution () 20 1.7 (1.76 1.70) 20 1.55 (1.61 1.55) 20 1.6 (1.66 1.60) 20 0.9 R sym a (%) 3.7 (9.4) 4.8 (2.8) 4.4 (3. 1) 8.2 28.1( 12.8 ) 28.1 (4.9) 3 5. 9(5 .8) 22.4 Completeness (%) 96.7 (98.1) 90.0 (85.4) 90.7 (9 6.9) 92.3 Average Redund ancy 3.3 (3.2) 4.6 (4.6) 3.2 (3.1) 5.9 Number of unique reflections 26,105 31,566 29,306 149,7 67 R cryst b /R free c (%) 16.0/19.1 16.2/19.3 17.9 / 21.1 10.6/1 3.1 No. atoms Protein 2 090 2 120 2 062 2 237 Water 287 248 288 546 B factors ( 2 ) main, side chain, solvent 13.2, 17.4, 24.8 13.7,17.1, 24.3 13.3/1 6.6 8.4/11 .3,31.9 R.M.S.D. 0.006/1.092 0.006/1.1 27 0.006 .014/. 031 Ramachandra n statistics (%) Most favored, allowed, outlier 87.5, 12.5, 0.0 88.5, 11.5, 0.0 87.1, 12.9, 0.0 87.1, 12.9, 0.0 a R sym b R cryst c R free is calculated in same manner as b Rcryst, except that it uses 5% of the reflection data omitted from refinement. *Values in parenthesis represent highest resolution bin.

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99 Table 4 2 Maximal values of rate constants for the hydration of CO 2 and dehyd ration of bicarbonate catalyzed by variants of H CAII and related pK a values, obtained by 18 O exchange at 25 C Variant k cat exch /K eff CO2 M 1 s 1 ) pK ZnH2O a k B 1 ) pK ZnH2O b pK donor b Wild type c 120 6.9 0.8 6.8 7.2 V143A 65 6 d 6.8 1.4 0.3 d 6.6 6.6 V143I 7.1 0.2 6.6 0.4 0.1 6.4 6.7 V143L 14 1 6.7 0.7 .04 6.5 6.5 a Obtained from the pH profile of k cat exch /K eff CO2 The st andard errors here are 0.1. b Obtained from the pH profiles of R H2O The standard errors here are 0.1 to 0.2. c From Fisher et al. ( 39 ) d Standard errors were determined from 18 O exchange data

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100 Table 4 3. Steady state constants for catalysis of th e hydration of CO 2 by variants of H CAII obtained by stopped flow spectrophotometry at 25 C and pH 8.3 Variant k cat /K m 1 s 1 ) k cat 1 ) Wild type a 120 1.0 V143A 42 1 b 0.5 0.2 b V143I 9.3 0.3 0.7 0.1 V143L 5.5 0.1 1.1 0.3 a From Steiner et al. ( 93 ) b Standard errors were determined from fits of steady state data.

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101 Table 4 4. Interatomic dista nces in for CO 2 in H CAII and variants. Distances for CO 2 atoms are measured from substrate atoms to the oxygen atom of the zinc bound solvent Atoms of CO 2 Wild type a Wild type b V143I c C 2.8 2.8 2.5 O1 3 2.8 2.8 O2 3.1 3.1 2.7 a From Do msic et al. ( 38 ) b From Sjoblom et al. ( 43 ) c This work. The uncertainty in these distances is 0.1

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102 Figure 4 1 pH profiles of k cat ex /K eff CO2 1 s 1 ) for t he hydration of CO 2 catalyzed by variants of H CAII ); and 18 O from CO 2 measured by membrane inlet mass spectrometry at 25 C in solutions c ontaining 25 mM 18 O enriched CO 2 /bicarbonate. No buffers were added

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103 Figure 4 2. pH profiles of RH 2 O/[E] (s 1) for the hydration of CO 2 catalyzed by the following variants of H CAII ); and V143L (green from CO 2 measured by membrane inlet mass spectrometry at 25 C in solutions containing 25 mM 18O enriched CO 2 /bicarbonate. No buffers were added

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104 A B Figure 4 3. Crystal structure at the active s ite of the A) V143I B) V143A and C) V143L HCAII variants Amino acids are as labeled. Only F o F c electron density map (blue) are shown for the 143 position. Solvent molecules are depicted as red spheres and the three histidines coordinating the zinc (dark gray). Figure was made using PyMol

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105 C Figure 4 3. Continued

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106 Figure 4 4. Stick overlay of position 143 H CAII structures. Wild type (orange), V143A (pink), V143L (yellow), and V143I HCAII (white). Amino acids are as labeled. In addition the three histi dines coordinating the zinc (dark gray) are shown. R.M.S.D. was 0.2 Figure was made using PyMol

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107 Figure 4 5. Stick stereo figure of the active site of V143I H CAII CO 2 /bicarbonate complex. Shown are the calculated omit Fo Fc electron density map, cont oured at 3 (green), when the A) CO 2 and B) bicarbonate were not incorporated into the respective models. Amino acids are as labeled. This figure was made using PyMol

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108 Figure 4 6. Structure of substrate/product bound in HCAII V143I. A) CO 2 and H 2 O molecules (red sphere), B) HCO 3 and C) as observed in the crystal structure, with dual occupancy (Refer to Figure 5). Amino acids are as labeled. This figure was made with PyMol

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109 Figure 4 7. Comparison of H CAII V143I substrate and product binding in H CAII Structur al overlay A) of CO 2 binding in wild type (green) (6) and V143I (yellow) HCAII, B) of bicarbonate binding in H200T (green) ( 117 ) and V143I (yellow). Amino acids are as labeled. This figure was made with PyMol

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110 CHAPTER 5 SOLVENT NETWORK, PRO TON TRANSFER A ND MARCUS THEORY In this chapter a variant Y7F N67Q H CAII that has an unbranched w ater network and in addition a shorter distance between His64 and the zinc bound solvent determined from the crystal structure at 1.6 resolution is discussed For this doub le mutant the 1 is ten fold greater than wild type measured by 18 O exchange. This allows a more complete understanding of the factors that influence proton transfer through water chains in a protein environment H CAII serves as a simple model for examining such proton transfers in more complex systems such as the photosynthetic reaction center, bacteriorhodopsin, and ATP synthase. This chapter also discusses HCAII as it applies to Marcus Theory to understand ca talytic proton transfer in the biophysical context of other work. Although devised for electron transfer, this approach has been applied to proton transfers ( 19 93 ) and specifically to proton transfer during catalysis by carbonic anhydrase ( 98 ) The proton transfer in catalysis by H CAII is characterized by highly curved Marcus (free energy) plots s uggestive of an inverted region, a region in which the rate constant for proton a (difference between donor and acceptor pK a ) becomes more favorable ( increases, an extensio n of previous work ( 1, 6,93 ) to studies examining deuteron transfer and to a new variant of H CAII that suggests an inverted region in the free energy plot. Inverte d regions in proton transfer reactions have been reported previously ( 11, 94 ) The The work presented on proton transfer and the solvent network is in strong collaboration with Dr. Rose Mikulski

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111 theoretical basis for these observations of inverted regions of a free energy plot has undergone much discussion; the interpretation of such an inverted region in catalysis by carbonic anhydrase implies a rapid, nearly barrierless, proton transfer through a preformed solvent network. Methods Site d irected Mutagenesis Please s ee Chapter 2 sections Site Directed Mutagenesis, Protein Expre ssion, and Affinity Chromatography for more information. 18 O Exchange Oxygen 18 experiments were performed as described in Chapter 2. With the addition of derivatives of imidazole and pyridine for Marcus Theory experiments at concentrations up to 200 mM we have observed a weak inhibition of both R 1 and R H 2 O This is probably due to binding at or in the vicinity of the zinc in the manner found for the binding of imidazole to carbonic anhydrase I ( 49 ) or 4 MI to a mutant of H CAII ( 9 5 ) The binding constant K i for this inhib ition is generally greater than 100 mM, indicating weak binding at the inhibitory site. Some exogenous donors exhibited no inhibition. In each case of inhibition, a single value K i described inhibition of both R 1 and R H2O as determined by these equations: R 1 obs = R 1 /(1+[B]/K i ) and R H 2 O obs = R H 2 O /(1+[B]/K i ). Stopped flow S pectrophotometry Measurements of initial velocity in the hydration of CO 2 and dehydration of bicarbonate were carried out on a stopped flow spectrophotometer (Applied Photophysics SX18.M V) using the initial 5% to 10% of the progress curve using the method of Khalifah ( 61 ) The hydration experiments were performed at pH 8.4 using 0.1

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112 HES M ES l red at 10 C. In all experiments, the ionic strength was maintained at 0.2 M using sodium sulfate. Saturated solutions of CO 2 were prepared by bubbling CO 2 gas into water at 25.0 C (saturating concentration 33.8 mM) and diluting using two coupled, air t ight syringes. Crystallography Crystals of the mutants N67Q and Y7F N67Q H CAII were obtained using the hanging drop method ( 83 ) protein [concentration ~15 mg/mL in 100 mM Tris precipitant solution [1.25 M sodium citrate 100 mM Tris Cl (pH 8.0)] against a well of t 20 C. Crystals were observed within a week of the crystallization setup at 293K. The N67Q and Y7F N67Q HCAII crystals were flash cooled using a 30% glycerol cryo protectant precipitant solution before mounting. The x ray data obtained at 100K, using an R AXIS V++ optic system from V arimax HR a Rigaku RU H3R Cu rotating anode operating at 50 kV and 100 mA. The detector to crystal distance was set to 80 mm. The oscillation steps were 1 with a 6 min exposure per image for 360 degrees. Data set statistics for the crystals are given in Table 1. The model building was done manually with the program Coot and refinement was carried out with P HENIX suite ( 86 ) The wild type H CAII structure, Fisher et al. ( 96 ) (PDB code: 1TBT ) with the waters removed and both His64 and Asn 67 residues mutated to Ala, was used as the starting phasing model.

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113 Results Catalysis The pH dependence of catalysis of the hydration of CO 2 determined by 18 O exch ange was determined for each of the variants of H CAII in Table 5 2. The data for Y7F H CAII were reported previously ( 57 ) These values are similar with values 1 s 1 The values of the pK a of the zinc bound water, pK a ZnH2O (Table 5 3, column 2) are also quite similar. These similarities reflect the large distance 7 to 10 between these side chains of residues 7 and 67 from the catalytic zinc The range in values of k B was nearly ten fold (Table 5 2). Catalysis by Y7F N67Q H CAII was measured by stopped flow spectroph otometry in the hydration and dehydration directions and compared with values for wild type. The steady state constants for these two enzymes were rather equivalent (Table 5 4). The solvent H/D kinetic isotope effect was determined for the turnover number for hydration, designated D (k cat ) hydration catalyzed by Y7F N67Q H CAII This value determined at pH 8.4 and 25 C was D (k cat ) hydration = 3.1 0.3. Crystal structures The crystal structures of the variants N67Q and Y7F N67Q of HCAII were solved to 1.5 1.6 resolution (Figures 5 1, 5 2). The final statistics of the structures including are listed in Table 5 1. No major structural perturbations were observed; the r.m.s.d. for C atoms was 0.1 for both variants when compared to wild type H CAII (PDB code 2 ILI ( 45 ) ). In H CAII His64 occupies an inward and outward conformation with respect to orientation in the active site cavity ( 9 7 98 ) The outward conformation was dominant in N67Q H CAII while the inward conformation w as observed in Y7F N67Q H CAII similar to the Y7F variant previously published ( 57)

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114 The hydrogen bonded solvent network in the active site cavity depicted in Figures 5 1 and 5 2 was conserved when compared with wild type. In the case of Y7F N67Q H CAII which has the most efficient rate constant for proton transfer, the apparent hydrogen bond between W2 and W3A, as well as that between W2 and W3B is longer in comparison with wild type (Table 5 5). However, the distance between W2 and N of His64 is shorter (Table 5 5). The carboxamide group of the glutamine substitution at residue 67 extended further into the active site cavity than the Asn residue it replaced. An associated effect was an increase by about 0.5 compared with wild type in the distance from the zinc bound solvent to the N of His64 the proton shuttle residue in its out conformation in N67Q (Table 5 5). The rate constant R H 2 O /[E] describes the release of H 2 18 O from the enzyme in the dehydration direction (eq 4) and is the component of the 18 O exchange that is dependent on proton transfer. For the site specific mutants H64G H CAII and N62L H64A N67L H CAII derivatives of imidazole and pyridine activated catalysis as measured by the exchange of 18 O between CO 2 and water. Thes e exogenous proton donors were found to increase R H 2 O /[E] in a saturable manner, shown for the activation of H64G H CAII by 4 methylimidazole (4 MI) in Figure 5 3. Such activation by 4 MI has been reported earlier for H64A H CAII ( 95 ) and for H64W H CAII ( 99 ) The fit of these data to the Michaelis form of eq 6 allowed estimates of the maximal rate constant for activation k B obs Data for activation of H64G H CAII by 4 MI demonstrate the gre ater enhancement of catalysis in H 2 O compared with (98%) D 2 O (Figure 5 3). These activations typically showed a solvent H/D isotope effect near 2.5 (see legend to Figure 5 3). The values of R H 2 O /[E] had not reached saturation at 200 mM of some proton

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115 donor s and had to be determined by extrapolation of activation plots to larger concentrations of proton donors. Also, in some cases this required that we take account of inhibition which was observed at higher levels of the derivatives of imidazole and pyridine The pH profiles of R H 2 O /[E] catalyzed by these variants are typically bell shaped reflecting proton transfer from the exogenous donor to the zinc bound hydroxide in the dehydration direction (eqs 2,4) Typical data are shown in Figure 5 4 for the activa tion of H64G H CAII in the presence of a saturating level, 100 mM, of the activator 4 MI. The data in Figure 5 4 for the values of R H 2 O /[E] in the presence 4 MI can be fit by eq 7 which describes activation as a single proton transfer between non interactin g donor and acceptor groups. This fit provides estimates of the maximal, pH independent value of k B the rate constant for proton transfer, and estimates of the values of the pK a of the donor and acceptor. This fit of the activation of H64G H CAII by 4 MI y ields values given in the legend to Figure 2 including a pK a of 6.8 0.1 for the zinc bound water and 8.4 0.1 for 4 methylimidazole. This is to be compared with the pK a of 7.2 0.1 measured for the zinc bound water determined from the pH profile of k c at exch /K eff CO2 catalyzed by H64G H CAII (Figure 5 5), and with the solution pK a of 7.9 for 4 MI. For many of the exogenous proton donors used here, the value of k B was determined using k B obs from activation data as in Figure 5 3, and inserting into eq 7 the solution value of (K a ) donor and the value of (K a ) ZnH2O obtained from the pH profile of k cat ex /K eff CO2 The pH profiles of k cat ex /K eff CO2 for hydration of CO 2 catalyzed by H64G H CAII and the triple mutant N62L H64A N67L H CAII (Figure 5 5) give an estimat e of the pK a for the zinc bound water molecule. Values of k cat ex /K eff CO2 are as great at 10 8 M 1 s 1 close

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116 to diffusion control. We sometimes observed a weak inhibition in k cat ex /K eff CO2 and R H 2 O /]E] caused by addition of imidazole and pyridine derivatives with values of the inhibition constant K i between 150 mM and 250 mM (data not shown). This effect has been described for H64A H CAII for the binding of 4 MI as a second sphere ligand of the zinc ( 100 ) and in Methods. Free energy plots Free energy plots were constructed for k B in catalysis by H64G H CAII in H 2 O and (98%) D 2 O (Figure 5 6), and for the triple mutant (Figure 5 7) These f ree energy plots show curvature of k B especially for the triple mutant in which the pK a of the zinc bound water is low at 6.1. This is lower than in wild type or H64G H CAII for which the pK a is near 7. What is notable about these data is that the lower pK a of the zinc bound water for N62L H64A N67L H CAII in effect shifts the free energy plot to the left (Figure 5 7); t his allows a more complete view of the curvature of the free energy pK a > 0.5 in Figure 5 7). Discussion Solvent Network and Proton Transfer These catalytic and structural results on variants of H CAII with amino acid replacements at residues in contact with water molecules in the active site cavity provide insight into t he proton transfer rates in this protein environment. The rate constant for proton transfer k B for N67Q Y7F H CAII 1 (Table 5 2) is the fastest measured for a variant of this isozyme, is derived from the 18 O exchange data, and represents proton transfer to the zinc bound hydroxide, presumably from the proton shuttle His64. The previously studied Y7F H CAII was also found to have rapid proton transfer at k B 1 ( 57 ) The value for wild type H CAII is k B 1 (Table 5 2) ( 38 )

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117 These high k B values observed for Y7F and N67Q Y7F H CAII were not explained by differences in the values of the pK a of the proton donor (His64) and acceptor (zinc bound hydroxide). This is shown in Table 3 in which the values of pK a are similar for these varian ts. Previous reports showed that the pH dependence of k B is rather flat in a (pK a ZnH2O pK a His64 ) near zero ( 1,6 ) When catalysis was measured by stopped flow spectrophotometry, the steady stat e constants k cat did not show the magnitude of differences between wild type and Y7F N67Q H CAII observed with k B from the 18 O exchange method (Table 5 4). The turnover number k cat for wild type H CAII is limited in rate by proton transfer at steady state, a s determined in part from the solvent H/D kinetic isotope effect D (k cat ) hydration of 3.8 ( 93, 1 01 ) When we measured D (k cat ) hydration for Y7F N67Q H CAII we obtained the somewhat lower v alue of 3.1 0.3. This is consistent with rate contributing steps for k cat in Y7F N67Q that do not involve proton transfer and are not present in k B which contains fewer steps of the catalysis. That is, the magnitude of k B is determined predominantly by proton transfer for both variants, while k cat involves steps from the enzyme substrate complex to the completion of the catalysis and may contain other steps which do not involve proton transfer such as product dissociation. Hence, we suggest that k B is th e best measure of proton transfer in this work. In the cases of Y7F and N67Q Y7F H CAII the enhanced rate constants are associated with an appreciable inward orientation of the His64 side chain (Figure 5 2). However, the orientation of His64 has not been sh own to affect the rate constant for proton transfer according to the following data. The variant N62L has His64 predominantly inward and N67L predominantly outward with other aspects of their

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118 protein structure nearly identical, yet their values of k B are t 1 ( 39 ) The same conclusion was reached using the mutant T200S ( 24 ) This is supported by computational studies which suggest that the orientation of His64 does not influence this intramolecular proton transfer rate ( 8 7 ) Although low ene rgy networks of ordered water are observed in crystal structures and are considered significant to understanding the proton transfer pathway, clusters of hydrogen bonded water in the active site cavity in solution have lifetimes typically in the picosecond range ( 108 ) The destruction of the water network caused by bulky, hydrophobic residues in the active site cavity is associated with substantial decrease in rate of proton transfer ( 39, 102 ) Comparison of the crystal structures of the variants of Table 5 1 shows that the two most efficient enzymes in proton transfer Y7F and Y7F N67Q H CAII have less branched water structure, specifically water molecule W3A is not observed in Y7F and is not within hydrogen bond distance of the chain in Y7F N67Q (Figure 5 2; Table 5 5). Computations show that proton transfer through an unbranched, hydrogen bonded water network is more rapid than through a branched pathway ( 47 103 ) This feature of a more direct pathway in the structures of Y7F and Y7F N67Q H CAII appears to be the most rel evant to explaining the proton transfer efficiencies of these variants. We also point out that this double mutant has the smallest distance among the variants examined here between the zinc bound solvent and N of His64 (Table 5 5). We attempted to demonstrate the enhanced proton transfer capacity of Y7F N67Q H CAII compared with wild type using stopped flow spectrophotometry in a steady state experiment. However, we were not able to demonstrate this due to the difficulties with

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119 buffers which are required to measure catalysis in the stopped flow experiment. We are constrained to use buffers that do not enter the active site cavity and exchange protons, buffers such as imidazole derivatives. We used buffers Ches a nd Mes which have a very limited capacity to enter the active site cavity but promote catalysis by exchange of buffers with His64. However, in order to demonstrate the enhance intramolecular rate of proton transfer in Y7F N67Q H CAII we need to enhance buf fer to the extent that this intramolecular step is strongly rate contributing. This means using substantial concentrations of the these buffers, say 100 mM, under which conditions the pH change during catalysis is small and rates are difficult to measure. This study with the greatly enhanced proton transfer of Y7F N67Q H CAII provides the clearest example to date of the relevance of the ordered water structure to rate constants for proton transfer. The complement of these studies with the pertinent computat ional results of rapid proton transfer through unbranched water chains ( 103, 104 ) is gratifying. It appears that this water structure as observed in crystal structures is a better predictor of efficient proton transf er than the orientation of the side chain of His 64. Marcus Theory To quantitate the extent of curvature of the free energy plots (Figure 4,5) and the observation of an inverted region, we use the Marcus theory for proton transfer (1 05 ) as applied to catalysis by carbonic an hydrase as described in earlier work (11 ) The observed activation barriers a re obtained from the pH independent, maximal values k B obs = RTln(hk B /kT), where h is the Planck constant and k is the Boltzmann a of the reactants, o = RTln[(K a ) acceptor /(K a ) d onor ]. In the Marcus approach, the observed overall activation

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120 obs is described in terms of the standard free energy of o o This approach is further modified to describe proton t ransfers in which there is a component of the observed activation o for the reaction. This component is called the work term w r for the forward direction (dehydration here) and w p for the reverse. In non enzym at ic, bimolec ular proton transfers, the work term is considered part of the free energy of reaction needed to bring the reactants together and form the reaction complex with associated solvation changes prior to proton transfer, although not necessarily associated with any distinct intermediate or energy minimum (28) The Marcus equation then becomes: obs = w r o obs w r + w p o ) 2 o ( 5 1 ) Here it is assumed that the work terms w r and w p as well as the intrinsic energy barri o do not vary for proton transfer between the series of homologous proton donors to which the equation is fit. This Marcus theory has been previously applied to variants of H CAII I (11 ) and H CAII ( 95 ) In Figure 5 6, the solid line describ es activation of proton transfer in the dehydration direction by imidazolium and pyridinium derivatives during catalysis by H64G H CAII The corresponding parameters for the fit are given in Table 2. The data o and large values of the work terms w r and w p consistent with previous results of similar experiments using different forms of carbonic anhydrase ( 11 95 ) The data indicate t hat the isotope effect on the o is unity (Table 2). This is also evident from the plots o would be evident

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121 in a greater difference in curvature between the data in H 2 O and D 2 O. A new feature of these data is the expression of the overall solvent H/D isotope effect, approximately 2.5, in the work terms although there is considerable propagation of error. This is contrary to the assumption that was made in our the previous interpretations of the free energy plot for proton transfer in carbonic anhydrase ( 11 ) where it was assumed that the H/D isotope effect was expressed in intrinsic kinetic barrier and not in the work terms; this assumption led to values of w r and w p that were not in agreement with values o btained using only H 2 O. The hint of an inverted region in Figure 5 4 appears supported in Figure 5 7 with a similar series of experiments using the triple mutant N62L H62A N67L H CAII This variant has the advantage that the pK a is 6.1 for the zinc bound w ater molecule, lower a as shown comparing Figures 5 and 4. The parameters of the Marcus equation are given in the legend to Figure 5 and are similar to those of H64G H CAII The very l o in Table 5 6 indicate a facile and nearly barrierless proton transfer, if this application of classical Marcus theory is applicable. These data are obtained for proton transfer from high concentrations of exogenous proton donors that we e stimate bind weakly at undetermined sites within the active site cavity. There is a barrier near 3 to 5 kcal/mol for thermoneutral proton transfer across a hydrogen bond of double potential o in Table 5 6 are consistent with proton transfer across a bond more closely related to a low barrier hydrogen bond. In fact, a short, strong hydrogen bond has been noted in the active site of H CAII (69 ) and its formation may be a necessary precursor to proton transfer. This

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122 may explain why the work terms are isotopically sensitive. This interpretation differs greatly from models that have been developed using comput ational meth ods which assign a barrier of 8 10 kcal/mol for the proton transfer in CA ( 103 106 ) Braun Sand et al. ( 106 ) have analyzed proton transf er in carbonic anhydrase using an empirical valence bond approach to a multi state model and conclude that the predominant barrier to catalysis is in the intrinsic kinetic barrier with rather small contributions from solvation factors. This gives an entire ly different interpretation of the free energy plots of Figure 4. Recent considerations of electron and proton transfer reactions suggest that an inverted region in a Marcus plot of proton transfer would not be accessible in the ranges of pH that are pract icable if only the driving force is altered (1 0 7 ) However, such inverted regions have been reported in experiments measuring rates of proton transfer (94, 108 ) and in this report (Figure 5 7), observations that have been difficult to interpret ( 1 10 ) An explanation of the inverted region in pr oton transfer is likely to include a dependence of other relevant parameters on the driving force of the reaction. Such parameters could be the vibrational frequencies of the proton and the distance between proton donor and acceptor (1 0 7) As a general consideration, the hydrogen bond distance between proton donor and acceptor in a bimolecular system is strongly influenced by the difference in pK a between them ( 109 ) Although the extent of curvature of the free energy plots of Figures 5 6 and 5 7may be enhanced by ancillary factors, the following is evidence that varying distances between proton donors and acceptors is not a major contributor. A prior study which determined the parameters of a fit of the Marcus equation to pr oton transfer in H CAII I

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123 ( 11 ) differs from the current study in that the proton transfer is intramolecular, from the proton shuttle residue His64 to the zinc bound hydroxyl in a series of variants with different values of pK a for the zinc bound water. This provides much less flexibility in distance between proton donor and acceptor. It is notable that the value of the intrinsic barrier for proton transfer in H CAII o = 1.4 0.3 kcal/mol, still very small, with values of the work function 6 10 kcal/mol ( 11 )

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124 Table 5 1. Crystal Structure Data and Refinement Statistics for N67Q and Y7F+N67Q H CAII Data collection statistics N67Q Y7F N67Q Wavelength () 0.9724 0.9724 Space group P 2 1 P 2 1 Unit cell paramet ers (,) a b c (); () 42.0, 41.2, 72.1; 104.3 42.1, 41.2, 71.9; 104.5 N umber of unique reflections Redundancy Completion % 35,820 3.6 (2.5) 97.7 (80.5) 31 729 3.8(3.6) 99.7 (99.8) Resolution () a R sym 50.0 1.50 (1.55 1.50) 7.3 (38.9) 14.5 (4.1) 50.0 1.60 (1.66 1.60) 8.4 (47.3) 13.1 (2.8) b R cryst (%) c R free (%) 18.5 21.4 16.6 20.5 Amino acid residues 3 261 3 261 No. of protein atoms 2180 2199 No. of H 2 O molecules 376 319 R.m.s.d. for bond lengths (), angles () 0.006 1.086 0.006 1 .019 Ramachandran statistics (%) Most favored, allowed, outlier 87.1, 12.4, 0.0 86.6, 13.4, 0.0 Average B factors ( 2 ) main side chain, Zn, solvent 18.2, 21.4, 11.5, 29.6 19.8, 22.0, 10.5, 28.7 a R sym 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. *Values in parenthesis represent highest resolution bin.

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125 Table 5 2 Maxi mal Values of Rate Constants for Hydration of CO 2 and Proton Transfer in Dehydration Catalyzed by H CAII and Variants Enzyme k cat exch /K eff CO2 CO 2 hydration 1 s 1 ) a k B proton transfer 1 ) b Wild type 120 0.8 Y7F c 120 3.9 N67Q 50 1.7 Y7F N67Q d 8 0 9.0 a Measured from the exchange of 18 O between CO 2 and water in the hydration direction (eq 5). Derived for each variant by a fit of the data (as in Figure 2) to a single ionization. The standard errors for these rate constants are generally 15% or le ss. b Measured from the exchange of 18 O between CO 2 and water using eq 6 in the dehydration direction. The standard errors are 22% or less. c Data at 10C from Fisher et al. ( 16 ) d Data at 10C.

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126 Table 5 3 Values of Apparent pK a Obtained by Kinetic Measurements of Catalysis by H CAII and Variants Enzyme pK a ZnH 2 O a pK a ZnH 2 O b pK a His64 b wild type 6.9 6.8 7.2 Y7F c 7.1 7.0 6.0 N67Q 6.5 6.7 6.6 Y7F N67Qd 6.9 6.3 6.2 a Measured from a fit of the data of Figure 2 to a single ionization. The standard errors in p K a are mostly 0.1 an d no greater than 0.2. b Measured from the fits of eq 6 to the data of Figure 3. The values of pK a have standard errors no greater than 0.2. c These data at 10 C from Fisher et al. ( 16 ) d These data at 10 C.

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127 Table 5 4. Steady state constants for the hydration of CO 2 and dehydration of bicarbonate catalyzed by wild type and Y7F N67Q H CAII a (k cat ) hydration 1 ) (k cat /K m ) hydration 1 s 1 ) (k cat ) dehyd 1 ) (k cat /K m ) dehyd 1 s 1 ) wild type H CAII 0.80 0.03 59 2 0.24 0.02 6.7 0.2 Y7F N67Q H CAII 0.67 0.04 67 4 0. 19 0.01 8.8 0.3 a The hydration experiments were performed at pH 8.4 and 25 C and the dehydration experiments at pH 6.0 and 10C.

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128 Table 5 5. Comparison of Distances () in the P roposed hydrogen B ond N etwork: Determined from the Crystal Structures of Variants of H CAII The error in these distances was 0.16 WT a Y7F b Y7F N67Q N67Q ZnSolvent W1 2.7 2.7 2.7 2.7 W1 W2 2.7 2.6 2.6 2.9 W2 W3a 2.8 n/a 4.3 2.9 W2 W3b 2.7 2.6 3.1 2.4 W2 H64(N ) 3.2/6.3 3.2 2.8 6.7 ZnSolvent H64(N ) 7.2/10.0 7.1 6.7 10.5 H64 in/out in in out a These data from (45), b These data from ( 16 )

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129 Figure 5 1. Structure of the active site of N67Q HCAII. Active s ite residues are shown as purple stick models. Thr199, Thr200, as well as t he three histid ine residues (His94, His96, His119) coordinating the zinc (gray sphere) are not labeled. The oxygen atoms of water molecules identified in the active site cavity are shown as red spheres Presumed hydrogen bonds are represented as dashed red lines. This fi gure was generated and rendered using Pymol

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130 Figure 5 2. A ctive site structures for Y7FY7F N67Q H CAII crystallized at pH 8.0. This diagram was constructed as described as in Figure 5

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131 Table 5 6 Parameters of the Marcus Equation for Activation of H64G H CAII by Exogenous Proton Donors in H 2 O and D 2 O (98%) a H2O D2O Solvent H/D Kinetic Isotope Effect b I o 0.21 + 0.04 0.18 + 0.06 0.95 + 0.11 w r 8.2 + 0.1 8.7 + 0.2 2.3 + 0.9 w p 8.2 + 0.1 8.6 + 0.2 2.0 + 0.7 a Conditions and proton donors as described i n Figure 2. b Solvent hydrogen isotope effect = exp[ I o /RT] or exp[ r /RT]

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132 Figure 5 3 2 2 O (98%) of R H2O ) by 4 methylimidazole in catalysis of 18 O exchange by H64G H CAII Solutions c ontained 25 mM of all species of CO 2 at pH 7.8 and 25 o C. The total ionic strength of solution was maintained at a minimum of 0.2 M by addition of Na 2 SO 4 The H/D kinetic isotope effect on the maximal value of R H2O /[E] is 2.3 0.2

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133 Figure 5 4 The depe ndence on pH of the rate constant R H2O /[E] (s 1 ) catalyzed by H64G H CAII and measured in the absence of 100 mM 4 type H CAII using solutions containing 25 mM of all species of CO 2 Ionic strength wa s maintained at a minimum of 0.2 M by addition of sodium sulfate. The solid line for H64G in the presence of 4 MI is a fit of eq 7 to the data with the rate constant for proton transfer k B 1 ; pK a ZnH2O = 6.8 0.1; and pK a 4 MI = 8.4 0.1 For wild type H CAII in the absence of buffer these values are k B 1 ; pK a ZnH2O = 6.8; pK a His64 = 7.2 (26 )

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134 Figure 5 5. Dependence on pH of k cat ex /K eff S for the hydration of CO 2 catalyzed by ( ) H64G H CAII H64A N67L H CAII H CAII Conditions were as described in Figure 2. Solid lines are fits to a single ionization with pK a of 7.2 0.1 for H64G; 6.1 0.1 for N62L H64A N67L H CAII ; and 6.8 0.1 for wild type H CAII

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135 Figure 5 B (s 1 ) for the activation of catalysis by H64G H CAII versus the difference in pK a values of the zinc bound water and the exogenous proton donor. Conditions were as described in Figure 1, except the pH of each experiment was at the pK a of the exogenous donor used. The following are the individual proton donors with their corresponding solution values of pK a under our conditions: a, 1,2 dimethylimidazole (pK a = 8.3); b, 2 methylimidazole (8.2); c, 2 ethylimidazole (8.0); d, 4 methylimidazole (7.9); e, imidazole (7.0); f, 3,4 dimethylpyridine (6.6); g, 4 methylpyridine (6.1); h, 3 methylpyridine (5.8); i, pyridine (5.3). The pK a of the zinc bound water molecule in H64G is pK a ZnH 2 O = 6.8 0.1. The solid lines are a fit of the Marcus equation to the data with parameters given in Table 2

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136 F igure 5 7. Values in H 2 O for k B (s 1 ) for the activation of catalysis by N62L H64A N67L H CAII versus the difference in pK a values of the zinc bound wat er and the exogenous proton donor. The added buffers are: a, 1,2 dimethylimidazole (pK a = 8.3); b, 2 methylimidazole (8.2); c, 4 methylimidazole (7.9); d, 1 methylimidazole (7.3); e, imidazole (7.0); f, 2,4 dimethylimidazole (6.9); g, 2,3 dimethylpyridine (6.6); h, 3,4 dimethylpyridine (6.6); i, 4 methylpyridine (6.1); j, 1 vinylimidazole (5.9); k, 3 methylpyridine (5.8); l, pyridine (5.3). The pK a of the zinc bound water in N62L H64A N67L H CAII is 6.1 0.1. The rate constants for proton transfer were dete rmined by 18O exchange from CO 2 water at 25 C in solutions containing the above buffers at ionic strength 2.0 M maintained with sodium sulfate. Total concentration of all CO 2 species was 0.1; wp = 6.6 0.5 kcal/mol)

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137 CHAPTER 6 FUTURE STUDIES HCAII in Environmental a nd Artificial Lung Research E mission of CO 2 has a severe impact on climate and is co nsidered a global pollution problem, with implications in global warming ( 1 15 ). To combat this problem, research has focused on biochemical fixation of CO 2 ( 1 16 ). This requires fast concentration and solubilization of CO 2 The capture of CO 2 collected i n the bioreactor is still challenging. Much work has focused on developing a model system that can efficiently concentrate CO 2 and remove it from the atmosphere. Science has look toward biotechnological methods to solve this problem. The catalytic effici ency of HCAII has made this enzyme a target for environmental research. Researchers have been considering HCAII as a component in bioreactors to aid in the removal of CO 2 from the atmosphere (11 2 11 3 ) One of the focuses of this research is attempting t o develop a more efficient version of HCAII. It has been shown that mutations in the CO 2 binding pocket of HCAII can impact CO 2 hydration ( 40 ). However, a HCAII variant in which catalytically efficiency has increased has yet to be discovered. Future work involves the discovery of a more efficient mutant of HCAII. Certain amino acid residues in the binding pocket of HCAII are implicated in the control of catalysis ( 40,41 ) and mutations in this region may promote faster hydration of CO 2 The goal would be to make mutations in the binding pocket that result in increased catalytic efficiency. Research discussed in Chapter 4 in corroboration with previous data (40) speculates that mutations at position 143 may not effective in increasing efficiency. Size an d hydrophobicity of substituted residues as well as conformational manipulations

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138 resulting from substituted residues should also be considered. The more efficient variants can then be used in bioreactors for fast sequestration and concentration of CO 2 T his would require development of a practical production process in regards to expression and purification of large quantities of active HCAII, but development of a more efficient variant of HCAII is the fi r st step. While HCAII has been proposed for assi stance in global warming, it also has implications for artificial lung research. A key biological function of the lungs is removal of CO 2 HCAII is expressed in the lungs and it participates in this process ( 1 14 ). As a result, HCAII would be valuable in the development of improved artificial lung technology. Future works focuses on development of temperature stable mutants for use in artificial lungs. The instability of enzymes at various temperatures and conditions make the rmally stable HCAII essential These variants still maintain their high catalytic activity. Mutations to increase melting temperature, primarily cysteine mutations to promote disulfide bridge formation are a primary area of focus. These highly active and thermally stable variants of HCAII would be valuable for use in artificial lung development to assist in the removal of CO 2 from the body. HCAII and Proton Transfer The proton transfer mechanism is highly complex. This involves the movement of protons over long distances. The dis tance from the zinc bound solvent to His64 is around 7 Human Carbonic anhydrase II has a very well understood proton transfer mechanism and if further work is done to better understand proton transfer in this enzyme, the information attained could prov ide insight into the complex proton transfer mechanisms of other enzymes like bacteriorhodopsin and ATP synthase It could also

PAGE 139

139 prove beneficial as a mimic for proteins in the same family like HCAIX, a membrane protein that is difficult to solve by cryst allography but has the same proton transfer mechanism that is implicated in tumorigenesis (18 Figure 6 1) Future work involves developing HCAII variants with a linear solvent network. Data from Y7F and Y7F/N67Q HCAII indicate faster proton transfer thr ough a more line ar solvent network. Further mutagenesis in the hydrophilic region should be focused on discovering residues that will promote removal of branched water molecules W3A and W3B (discussed in Chapter 5). The proton transfer rates would be mea sured and the impact of linearity would be deduced based on the fold changes compared to other variants. Development of a linear solvent network in HCAII will help to validate the claim that a linear water network would promote faster proton transfer, or prompt the consideration of other aspects, like pK a or the distance between the proton shuttle and solvent network, that can also contribute to the complex nature of proton transfer.

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14 0 Figure 6 1. Overlay of wild type HCAII (pink PDB ID: 2ILI ) and HCAIX mimic (yellow). The zinc atom is represented as a gray sphere and the bound water/hydroxide by a red sphere. The r.m.s.d. is 0.24 Figure was made using PyMol

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141 APPENDIX SEQUENCE ALIGNMENT O F MAMMALIAN CAS

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142 Figure A 1. Sequence a lignment of mammali an CAs

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143 Figure A 1. C ontinued

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144 Figure A 1. Continued

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145 Figure A 1. Continued

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146 LIST OF REFERENCES 1. Silverman DN McKenna R. Solvent Mediated Proton Transfer in Catalysis by Carbonic Anhydrase. Acc Chem Res 2007;40:669 675. 2. Bertini I, Luchinat C. The st ructure of cobalt(II) substituted carbonic anhydrase and its implications for the catalytic mechanism of the enzyme. Ann N Y Acad Sci 1984;429:89 98. 3. Liljas A, Hkansson K, Jonsson BH, Xue Y. Inhibition and catalysis of carbonic anhydrase. Recent crystallo graphic analyses. Eur J Biochem 1994;219(1 2):1 10. 4. Tu CK, Silverman DN. Catalysis by cobalt(II) substituted carbonic anhydrase II of the exchange of oxygen 18 between CO2 and H2O. Biochemistry 1985;24(21):5881 7. 5. Xu Y, Feng L, Jeffrey PD, Shi Y, Morel FM. Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms. Nature 2008;452(7183):56 61. 6. Lindskog S. Structure and mechanism of carbonic anhydrase. Pharmacol Ther 1997;74(1):1 20. 7. Whittington DA, Waheed A, Ulmasov B, Shah GN, Grubb J H, Sly WS, Christianson DW. Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells. Proc Natl Acad Sci U S A 2001;98(17):9545 50. 8. Vargas LA, Alvarez BV. Carbonic anhydrase XIV in the normal and hypertrophic myocardium. J Mol Cell Cardiol 2012;52(3):741 52. 9. Chegwidden WR, Carter ND and Edwards YH. The Carbonic Anhydrases New Horizons, Birkhauser Verlag, Basel 2000. 10. Supuran CT, Scozzafava A and Conway J. Car bonic Anhydrase Its Inhibitors and Activators, CRC Press, Boca Raton 2004. 11. Silverman DN, Tu C, Chen X, Tanhauser SM, Kresge AJ, Laipis PJ. Rate equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III. Biochemistry 1993;32 (40):10757 62. 12. Picaud SS, Muniz JR, Kramm A, Pilka ES, Kochan G, Oppermann U, Yue WW. Crystal structure of human carbonic anhydrase related protein VIII reveals the basis for catalytic silencing. Proteins 2009;76(2):507 11. 13. Pastorekova S, Parkkila S, Pasto rek J, Supuran CT. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem 2004;19(3):199 229.

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156 BIOGRAPHICAL SKETCH Dayne West was born in Melbourne, Florida on Thanksgiving. He was pretty muc and 280 pounds, he does not have the typical look of scientist, which he credits his years as football in player in high school and college. He graduated from Gaither High School in Tampa, Florida in 2004, ranked 23 out of 505 students in his graduating class, and 49 out of the 50 top high school football players in the state of Florida. He went to Delaware State University (DSU) in Dover, Delaware on a full football scholarship. Tragedy struck him with a knee injury severe enough to end his athletic career but not his academic one. He graduated summa cum laude from DSU in 2008 with a Bachelor of Science degree in chemistry. That same year he was accepted into gradu ate school at the University of Florida. He has worked under the mentorship of Dr. Robert McKenna. With his hard work, motivation and good karma he was able to graduate in four years, one year ahead of the 5 year graduate program average. He has seen immense scientific success and accolades. He has presented at numerous national conferences and has been awarded pre doctoral fellowships from the National Science Foundation, the National Institutes of Health and the University of Florida. In his four year career at UF, he will have published 5 papers in various journals. Outside of research, he has been an ambassador for the promotion of minority students in science. He has attended the Annual Biomedical Research Conference for Minority S tudents (ABRCMS) as a recruiter for the University of Florida. He hopes to have a long, successful scientific care er