KINETICS AND STRUCTURE OF PROTON TRANSFER PATHWAYS IN CARBONIC ANHYDRASE

By

ROSE LYNN MIKULSKI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

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© 2010 Rose Lynn Mikulski

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To Christopher, Geralyn, Jasmine, Michael, and Marigold

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ACKNOWLEDGMENTS

I thank my mentor Dr. David Silverman for his endless support of my pursuits over

these year. Dr. Silverman exposed me to the many facets of academia and allowed me

to explore my interests ensuring that I have the confidence in myself as a scientist to

pursue the career of my choice. I thank my co-chair Dr. Robert McKenna for his

mentoring and enduring enthusiasm. I would also like to thank my other committee

members, Dr. William Kem and Dr. Susan Frost. My committee supported me with honest

critiques and challenging inquisitiveness to direct a successful path to my PhD.

I also sincerely thank Dr. Chingkuang Tu for his patience and sharing his

tremendous knowledge of enzyme kinetics, and general biochemistry me with. I would

like to thank Dr. Mavis Agbandje-McKenna, Deepa Bhatt, Patrick Quint, Zoe Fisher, and

John Domsic for all their assistance, and advice. The graduate students Nicolette Case,

Katherine Sipple, Balu Avaur, Dayne West, Balasubramanian Venkatakrishnan, Mayank

Aggarwal, Ha-Long Mguyen, Joannalyn Delacruz, Erin Mack-Humphrey, and Karlie

Bonstaff have been sounding boards, sources of advice and expertise and I thank them for

being my friends. I would also like to thank Wayne McCormack for improving the program

and being available to talk during the successes and struggles. I thank the ladies who run

the pharmacology department for taking care of the bureaucratic ins and outs for me.

Lastly, I would like to thank my family to whom this is dedicated. My younger siblings

have been a source of escape from the stress, words of encouragement, and the best

medicine in the world laughter. I thank my mother and father for fostering my curious nature,

helping me to examine scientifically the world around me from a young age, and for

inspiring me to succeed and do what I love.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS.................................................................................................. 4 

LIST OF TABLES............................................................................................................ 7 

LIST OF FIGURES.......................................................................................................... 8 

LIST OF ABBREVIATIONS........................................................................................... 10 

ABSTRACT ................................................................................................................... 14

CHAPTER

1 INTRODUCTION .................................................................................................... 16 

The Carbonic Anhdrases ........................................................................................ 16 Catalytic Mechanism............................................................................................... 17 Structure ................................................................................................................. 18 

Solvated Active Site ......................................................................................... 19 Proton Shuttle Residue .................................................................................... 20 Extended Active Site ........................................................................................ 21 

Speeding Up Proton Transfer ................................................................................. 22 Physiological and Medical Significance. ................................................................. 23 

2 KINETICS OF SPEEDING UP PROTON TRANSFER IN HUMAN CARBONIC ANHYDRASE II ...................................................................................................... 27 

Extended Active Site Hydrophilic Residues ............................................................ 27 Faster Rates of Proton Transfer ............................................................................. 28 Methods .................................................................................................................. 29 

Expression and Purification of Enzymes .......................................................... 29 Kinetics............................................................................................................. 29 18O Exchange................................................................................................... 29 Esterase Activity............................................................................................... 31 

Results.................................................................................................................... 31 Discussion .............................................................................................................. 33 

3 STRUCTURE OF PROTON TRANSFER PATHWAYS IN HUMAN CARBONIC ANHYDRASE II ...................................................................................................... 46 

Structural Role of Water Network in Proton Transfer .............................................. 46 Proton Shuttle Residue........................................................................................... 46 Methods .................................................................................................................. 47 

X-Ray Crystallography...................................................................................... 47 Thermodynamic Stability .................................................................................. 49 

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Results.................................................................................................................... 49 Structure of Y7I HCA II ..................................................................................... 49 Role of Tyr7 in Thermal Stability of HCA II ....................................................... 51 Structures of N62Q, N67Q and Y7F+N67Q HCA II .......................................... 51 

Discussion .............................................................................................................. 52 

4 PHYSIOLOGIAL ROLE OF HUMAN RED BLOOD CELLS IN THE GENERATION OF NITRIC OXIDE ......................................................................... 66 

Generation of NO Catalyzed by CA ........................................................................ 67 Deoxy-Hemoglobin Mechanism of Nitrite Reductase.............................................. 68 Methods .................................................................................................................. 69 

Materials........................................................................................................... 69 Inlet probe and Kinetic Measurements ............................................................. 70 

Results.................................................................................................................... 71 NO Generation from CA ................................................................................... 71 Deoxy-Hemoglobin Catalyzed Generation of NO............................................. 72 Human Red Blood Cell Suspensions Generation of NO from Nitrite ................ 73 Effect of EAZ on NO Accumulation .................................................................. 75 Effect of DIDS on NO Accumulation................................................................. 75 Effect of PCMBS on NO Accumulation............................................................. 76 

Discussion .............................................................................................................. 77 Accumulation of Extracellular NO in Red Cell Suspensions ............................. 77 Inhibition of Band 3 Anion Exchanger of Red Cells .......................................... 80 Inhibition of Aquaporin-1 Channel of Red Cells................................................ 81 

5 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................... 91 

LIST OF REFERENCES ............................................................................................... 96 

BIOGRAPHICAL SKETCH.......................................................................................... 104 

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LIST OF TABLES

Table page 2-1 Kinetic rate constants for HCA II and mutants. ................................................... 36 

2-2 Values of apparent pKa obtained by various kinetic measurements of catalysis in HCA II and mutants.......................................................................... 37 

3-1 Dataset, refinement, and final model statistics for the crystallographic study of N62Q, N67Q HCA II, N67Q+Y7F HCA II, and Y7I HCA II .............................. 57 

3-2 Thermodynamics of unfolding of wild type and Y7 variants of HCA II. a

Calorimetric parameters determined by DSC. .................................................... 58 

3-3 Comparison of proposed hydrogen bond networks for wild type, Y7F, Y7F+N67Q, N67Q, N62Q, and Y7I HCA II. ........................................................ 59 

3-4 Comparison of the features proposed to regulate proton transfer rates in the protein environment in HCA II and selected variants.......................................... 60 

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LIST OF FIGURES

Figure page 1-1 The active site of HCA II. .................................................................................... 25 

2-1 The pH profiles for kcatex/Keff

CO2 (M-1s-1) for the hydration of CO2 catalyzed by HCA II and hydrophobic Tyr7 substitutions. ....................................................... 38 

2-2 The pH profiles for kcatex/Keff

CO2 (M-1s-1) for the hydration of CO2 catalyzed by HCA II and polar substitutions at Tyr7. ............................................................... 39 

2-3 The pH profiles for kcatex/Keff

CO2 (M-1s-1) for the hydration of CO2 catalyzed by HCA II and conservative substitutions at Tyr7, Asn62 and Asn67...................... 40 

2-4 The pH profiles for the proton-transfer dependent rate of release of 18O-labeled water by HCA II and hydrophobic substitutions...................................... 41 

2-5 The pH profiles for the proton-transfer dependent rate of release of 18O-labeled water by HCA II polar substitutions at Tyr7. ........................................... 42 

2-6 The pH profiles for the proton-transfer dependent rate of release of 18O-labeled water by HCA II and conservative substitutions at Tyr7, Asn62 and Asn67 ................................................................................................................. 43 

2-7 Free energy plot of proton transfer in HCA II and variants. ................................ 44 

2-8 Double mutant catalytic cycle . ........................................................................... 45 

3-1 Crystal structures of the active sites of Y7I and Y7F HCA II............................... 61 

3-2 Overall comparison of wild-type HCA II and Y7I HCA II ..................................... 62 

3-3 Representation of crystal contacts in Y7I HCA II. ............................................... 63 

3-4 Differential scanning calorimetry profiles for wt HCA II and mutants.. ................ 64 

3-5 Crystal structures of the active sites of N62Q and N67Q and Y7F+N67Q HCA II. ................................................................................................................ 65 

4-1 NO accumulation from nitrite at various concentrations of HCA II. ..................... 82 

4-2 NO accumulation from various globular proteins and metal ion contamination. . 83 

4-3 The time course of NO accumulation from various forms of hemoglobin. .......... 84 

4-4 Extracellular NO accumulation obtained from a suspension of degassed human red cells.. ................................................................................................ 85 

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4-5 The pH dependence of extracellular NO accumulation. ..................................... 86 

4-6 The dependence on hematocrit of extracellular NO accumulation.. ................... 87 

4-7 The effect of EZA on extracellular NO by degassed human erythrocytes. ......... 88 

4-8 The effect of DIDS on extracellular NO by degassed human erythrocytes......... 89 

4-9 The effect of PCMBS on extracellular NO by degassed human erythrocytes..... 90 

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LIST OF ABBREVIATIONS

Å Angstrom

A alanine

AE anion exchange

ACZ acetazolamideatm

ACES N-(2-Acetamido)-2-aminoethanesulfonic acid

BH+ protonated base

BSA bovine serum albumin

C Celsius

CAM carbonic anhydrase from Methanosarcina thermophila

CA carbonic anhydrase

CAPS N-cyclohexyl-3-aminopropanesulfonic acid

CHES N-cyclohexyl-2-aminoethanesulfonic acid

Cl chloride

cm centimeter

CO2 carbon dioxide

ΔCp change in eat capacity

° degree

D aspartic acid

deoxy deoxygenated

DIDS 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid

DNA deoxyribonucleic acid

DNDs 4,4'-dinitrostilbene-2,2'-disulfonic acid, disodium salt

DTPA diethylenetriaminepentaacetic acid

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DSC differential scanning calorimetry

E enzyme

E. coli Escherichia coli

EDTA ethylenediaminetetraacetic acid

eNOS endothelial nitric oxide synthase

EPR electron paramagnetic resonance

eV electron volt

EZA ethoxyzolamide

F phenlyalanine

Fe2+ Iron

GI gastrointestinal

ΔG Gibbs free energy

H histidine

H+ proton / hydrogen ion

Hb(FeII) hemoglobin ferrous iron

HCT hematocrit

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

His64 proton shuttle residue histidine 64 in HCA II

HCA human carbonic anhydrase

HCO3 bicarbonate ion

ΔH°m melting enthalpy

I isoleucine

IC50 concentration required for 50% inhibition

IPTG isopropyl-β-D-thiogalactopyranoside

K rate constant

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kB rate constant for proton transfer

kcal kilocalorie

kcat turnover number

KM Michaelis constant

kcat/KM specificity constant

kDa kilodalton

kcatexch/Keff

CO2 specificity constant determined for hydration of CO2 by CA

LB luria broth

M molar

MES 2-(4-morpholino)-ethane sulfonic acid

Met reduced to ferric iron

MOPS 3-(N-morpholino)-propanesulfonic acid

μM micromolar

mM millimolar

mA milliamp

mol mole

m/z mass to charge ratio

N asparagines

nM nanomolar

NO nitric oxide

N2O3 dinitrogen trioxide

18O stable isotope of oxygen with atomic mass of 18

OD optical density

OH- hydroxide ion

PCMBS Para-chloromercuribenzene sulfonic acid, sodium salt

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PDB Protein Data Bank

pH negative log of proton concentration

pKa acid dissociation constant

Q glutamine

R arginine

rmsd root mean square deviation

RT room temperature

S serine

SITS 4-acetamido-4’-isothiocyanstilbene-2,2’-disulfonic acid

disodium salt

TAPS N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid

Tm temperature melting

Tris tris(hydroxymethyl)aminomethane

torr Torr (unit of pressure)

W tryptophan

wt wild type

Zn zinc

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

KINETICS AND STRUCTURE OF PROTON TRANSFER PATHWAYS

IN CARBONIC ANHYDRASE

By

Rose Lynn Mikulski

December 2010

Chair: David Silverman Major: Medical Sciences – Physiology and Pharmacology

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the

reversible hydration of carbon dioxide to bicarbonate and a proton. Catalysis involves

an intramolecular proton transfer that delivers an excess proton from the zinc-bound

water to an internal proton acceptor, His64. His64 shuttles this proton to the bulk

solvent, thus regenerating the active site for the next catalysis. The ability to

experimentally increase the rate of proton transfer within the HCA II active site can

provide insight into the biophysical properties for this process. The three factors

proposed to influence the rate limiting step of proton transfer (kB) are the active site

water network, conformation of His64, and the pKa of both the zinc bound solvent and

His64.

An extensive analysis of the kinetic and structure of wild type and several mutants

of HCA II were conducted over a broad pH range. The results show that the enzyme

active site is very stable. Several mutants altered the proton shuttle His64 orientation,

the water network, and the pKa of the defined proton donor and acceptor which resulted

in altered proton transfer rates. Faster rates of proton transfer were observed in all the

mutants. The 2-4-fold increases in proton transfer followed the theoretical values

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predicated by Marcus theory based on changes in the pKa. The 7-15 fold increase in

proton transfer over wild type showed residue His64 primarily in the inward

conformation decreasing the distance to the zinc. The less branched water network with

more conventional hydrogen bonds lengths connecting zinc solvent through only two

water molecules to His64 appeared in mutants with enhanced proton transfer rates

compared to wild type HCA II.

Classically the physiological role of CA is in acid-base balance throughout the

body. A recent proposal that CA could catalyze the conversion of nitrite into nitric oxide

(NO) a potent vasodilator prompted the design of a system to directly measure NO

concentrations in red cell suspensions. The examination of CA alone as well as CA

within whole RBC as well as hemoglobin alone did not show sufficient generation of NO

at physiological levels of nitrite for vasodilation.

CHAPTER 1 INTRODUCTION

The Carbonic Anhdrases

The carbonic anhydrases (CA)s are commonly characterized as zinc

metalloenzymes that rapidly catalyze the reversible hydration of carbon dioxide to form

bicarbonate and a proton [1]. The well studied classes of the enzyme are: α, β, and γ

[2]. The α-class was first discovered in and still predominantly found in mammals [3].

The β-class includes both plant and several bacterial CAs such as that of Escherichia

coli. Finally the γ-class consists of archaeal CAs. These three classes of CA have been

extensively studied kinetically and are believed to share a similar mechanism.

The classes of CA vary significantly in their structures which make them an

example of convergent evolution. The α CA’s contain one zinc per molecule, a

characteristic of the conserved active site coordination by three histidine residues and a

solvent molecule [2]. The α class are predominatly monomeric, near 30 kDa molecular

weight except human CA IX which has recently been shown to be a functional dimmer

of about 52-58 kDa [4, 5]. The β class shows the widest structural deviation with varying

oligermerization leading to molecular weights up to 200 kDa. The zinc coordination of

the β-class consists of one histidine and two cysteines residues, while the fourth

coordination site is occupied by a solvent molecule or an aspartate in type I and II

enzymes respectively. The γ-class are trimers with metal coordination analogous in

structure to the α CAs and in the case of the CAM enzymes contain iron in place of the

zinc under anaerobic growth[6].

The human genome is shown to code for at least 15 different isozymes of CA [7].

These isozymes vary in their expression, distribution, localization, and kinetic activity.

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The cytosolic isoforms are I, II, III, and VII, and include the mitochondrial enzymes VA

and VB. The extracellular isoforms include: the membrane bound enzymes by a

transmembrane domain (IX, XII and XIV) or GPI-anchor (IV and XV) as well as the only

secreted isoform VI. The isoforms VIII, X, and XI are considered CA-related proteins

because they are missing one or more of the conserved Zn coordinating histidine

residues rendering them catalytically dead.

Catalytic Mechanism

The catalytic conversion between carbon dioxide and bicarbonate by CA is a well

studied mechanism that is believed to be shared by all CA’s [1, 7, 8]. Human isozyme II

of CA is one of the most extensively studied and is very fast with catalytic turnover of

~1×106 s-1. A wide body of evidence supports a ping-pong mechanism with two steps

[1].

+ H2O EZnOH- + CO2 ⇋EZnHCO3

- ⇋ EZnH2O + HCO3- (1)

His64-EZnH2O + B ⇋ H+His64-EZnOH- + B ⇋ His64-EZnOH- + BH+ (2)

The first stage is the conversion of CO2 into HCO3- which is completely reversible. The

hydration direction begins when the catalytically active zinc-bound hydroxide

encounters a CO2 as shown in eq 1. The literature often describes this process as a

direct nucleophilic attack of the zinc-bound hydroxide on CO2. The CO2 may otherwise

interact with the zinc through a general base mechanism. A better understanding of the

mechanism can be obtained through solvent H/D isotope effects and the structure of a

bicarbonate-CA complex [9, 10]. The absence of an H/D isotope effect on kcat/Km for

the initial stage of the catalysis (eq 1) was confirmed by both 18O exchange [11] and 13C

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NMR [12]. The lack of a measurable isotope effect supports a direct nucleophilic attack

mechanism of the zinc-bound hydroxide [9, 13]. There does not appear to be a rate

influencing proton transfer event in this stage of the catalysis by CA [12].

The second stage of the catalytic mechanism involves the regeneration of the zinc

bound hydroxide. This step is also the rate limiting intramolecular proton transfer in HCA

II. The presence of an intramolecular proton shuttle in CA was predicted to be a side

chain such as histidine with a pKa near 7.0 similar to that of the zinc-bound water [14].

The ability to rapidly transfer the protons in the catalysis of CA is now attributed in part

to the side chain of His64 based on several important experiments [15]. Through site

directed mutagenesis of the human CAII protein, the effect of substituting the His64 with

an alanine reduced the rate of proton transfer (eq 2) up to 50-fold [16]. The rate of CO2

and HCO3- (eq 1) interconversion remained constant in the H64A mutant [16]. Further

supporting evidence were “chemical rescue” studies that show recovery of activity in the

H64A mutant upon the addition of imidazole [16]. The rate of the slower HCA III

isozyme can be increased by 10-fold by replacing the endogenous Lys64 with a

histidine [14]. The mutation to a histidine in HCA III also shows a kinetic profile including

solvent H/D isotope effects and pH-rate profiles more like HCA II [14]. These

experiments signify the extent to which CA’s active site can be tuned to carry out the

relatively simple hydrolysis of water.

Structure

The first crystal structure of HCA II was determined by Liljas and colleagues in

1972 and now there are more than 200 CA crystal structures in the protein data bank

[17]. Overall HCA II is a single-domain, globular protein that is almost spherical with

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approximate dimensions of 80 nm3 [18, 19]. The HCA II structure can be described as a

10-stranded twisted β-sheet, which is decorated on the surface by seven α−helices. The

strands of the β-sheet are mainly anti-parallel, with the exception of two pairs of parallel

strands. There is a conserved loop region extending towards the active site that

contains the proton shuttling residue, His64.

The active site is a conical cavity centrally located about 15 Å deep inside the

spherical protein (Figure 1-1) [17]. The catalytic zinc ion (Zn2+) is located at the bottom

of the cleft, with tetrahedral coordination by three histidine residues (94, 96, and 119)

and a highly polarized water molecule or hydroxyl group (Figure 1-1). The cavity

contains a region of hydrophobic residues (Val121, Val143, Leu198, Thr199, Val203

and Trp209) where the carbon dioxide is held in orientation to interact with the zinc [20].

Leading out of the cavity is a patch of more hydrophilic residues (Tyr7, Asn62, His64,

Ans67, Thr199 and Thr200). These hydrophilic residues are believed to help stabilize

an active site water network though hydrogen bonding.

Solvated Active Site

The ordered network between the zinc-bound solvent and His64 is depicted in the

crystal structure at 1.05 Å resolution (Figure 1-1) [19]. Several amino acids (Tyr7,

Asn62, Asn67, Thr199, and Thr200) appear close enough to participate in coordinating

a solvent network (W1, W2, W3a, and W3b). Thr199 is purported to hydrogen bond to

the zinc-bound solvent that, in turn, is hydrogen bonded to W1. Thr200 and the next

solvent in the chain, W2, further stabilize W1 through hydrogen bonds [9, 21]. The

solvent network then apparently branches as W2 hydrogen bonds to both W3a and

W3b. The hydroxyl group of Tyr7 further orientates the W3a, while Asn62 and Asn67

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stabilize W3b. This solvent network is conserved in numerous crystallographic

structures; which localizes W2, W3a, and W3b in close proximity to the side chain of

His64 (Figure 1-1)[19, 22, 23].

Examination of the structure of HCA II at near atomic resolution reveals aspects

not apparent in lower resolution studies [19, 22]. For instance, the solvent molecule W2

(the only ordered solvent molecule in the active site stabilized exclusively by other

solvent molecules) is trigonally coordinated with equal distance (2.75 Å) by W1, W3a,

and W3b. Only W2 is in the plane of the imidazole ring of His64 and within hydrogen-

bonding distance of the D1 nitrogen in the His64 imidazol ring when in the inward

conformation (Figures 1-1). A more recent study also examined a water molecule in the

hydrophobic region of the active site that forms a short strong hydrogen bond (2.45 Å)

to the zinc bound solvent [22]. This deep water may contribute to the catalysis through

providing an energy barrier to the binding of CO2 in the enzyme−substrate complex,

which can enhance the conversion rate of CO2 into bicarbonate, as well as to lower the

pKa of the zinc-bound water and to promote proton transfer.

Proton Shuttle Residue

Early structures of HCA II implicated His64 in intramolecular proton transfer

despite the ~7 Å distance separating it from the zinc [23]. A highly refined

crystallographic structure showing a water bridge between the zinc and His64 supported

the above suggestion of intramolecular proton transfer in HCA II [19]. Evidence of the

ability of His64 to help shuttle the protons from the active site out into the solution has

been strengthen by crystallographic analysis confirming the “in” and “out” or flip-flop of

the imidazole side chain of the histidine residue [19, 24]. The conformations of His64

are characterized by side chain torsion angle rotamers of 1 around the Cα-Cβ bond

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between the side chain and protein back bone. The inward orientation pointing towards

the zinc in the active site is defined by dihedral angles χ1 of 44° and of χ2 95°. The

outward orientation points toward the bulk solvent surrounding the surface of the protein

and is defined by dihedral angles of χ1 of -39° and χ2 of 98°. Other CA’s including the γ-

class CAM, show two rotamers of the proton transfer residue Glu84 residue [25].

Several orientations of a chemically modified cysteine residue in a mutant of murine CA

V are observed and implicated in proton transfer [26].

The occupancy of the H64 side chain in HCA II appears distributed between the

inward and outward conformation to transfer protons intramolecularly. The orientation of

the His64 has shown some pH dependence such that the inward conformation is

increasingly occupied in increasting pH from 6.0 to 9.0 in crystal structures [19]. The

Grotthuss mechanism explains that high proton mobility can be achieved in the active

site by protons hopping along the water network’s hydrogen bonds with minimized

movement of the oxygen atoms [27]. The migration of protons through the active site of

CA is highly interesting to theorists as it can be applied to other biologically important

proton transfers in proteins and their catalysis. Several computational labs have applied

theory to the simulation of proton transfers with CA as the model [27-31].

Extended Active Site

The activity of CA may start at the catalytic zinc but the entire environment allows

the efficient catalysis of the enzyme. Recent mutational studies in the active site of HCA

II show how other residues within the H64 environment modulate the flexibility of the

imidazole side chain altering the ability to transfer protons. Insight into the highly

evolved active site was gained through mutation of Tyr7, Asn62 and Asn67, which

surround the His64 on either side in the hydrophilic portion of the cavity [23, 32].

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Substitution of hydrophobic residues at these positions was shown to cause changes in

the rate of proton transfer, the orientation of His64, the pKa of His64, disruption of the

water network, and further steric changes to other residue side chains [18]. A typical

case is the mutagenesis of Asn67 to leucine in HCA II, which altered the solvent

structure, caused nearly complete “out” orientation of the His 64 side chain, and

decreased the proton transfer rate constant to 0.2 μs-1 [23]. A recent examination of

several hydrophobic mutations at position 62 showed maintained water networks and

concluded that a role of Asn62 in HCA II is to permit two conformations of the side chain

of His64 [32].

Speeding Up Proton Transfer

The difficulty in understanding the speed at which proton transfer occurs within

HCA II is predicated on accurate identification of the proton-conducting pathway. The

proposed intramolecular proton channel is ZnH2O through W1 to W2 and bridged from

W3a and W3b to the His64 and ultimately out to the bulk solvent (Figure 1-1).

Identifying the proton donor as the zinc bound solvent and His64 as the intramolecular

proton acceptor allows us to correlate changes in the electrostatics of the active site by

the ΔpKa (ΔpKa ZnH2O - ΔpKa His64). Future mutational analysis should help to confirm

this pathway and important residues that help maintain the electrostatics of the active

site. The mutation of Tyr7 into a non-polar hydrophobic phenylalanine exhibited a rate

constant for proton transfer seven-fold greater than the wild type HCA II [23]. The Y7F

substitution did not have an effect on the catalysis of the hydration-dehydration step

thus being consistent with wild type serves as a control and indicated that no

perturbations are caused at the Zn-OH/H2O ~7.0 Å away [23]. The kinetics also showed

that the His64 pKa was lowered from 7.0 in wild type to about 6.0. The more acidic side

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chain may correlate with a faster proton transfer in the dehydration direction.

Structurally, the Y7F HCA II differed in that His64 was mainly inward and the W3a was

missing. The absence of W3a possibly allows a more streamlined water network.

A simplified water wire leading to faster proton transfer agrees with in silico studies

where branched arrays form a less efficient “Eigen”-like solvent structures (H9O4+, that

is H3O+-3(H2O) [28]. The hydroxyl of Tyr7 appears to be within hydrogen bonding

distance of water molecule W3a. However, the recent neutron crystal structure recently

determined at pH 9 shows the tyrosine hydroxyl unprotonated and no such hydrogen

bond to solvent [33].

Physiological and Medical Significance.

Depending on where the particular CA resides it can be implicated in a number of

important physiological functions including: 1) the rapid conversion of HCO3- into CO2,

as in red cells and in photosynthesis; 2) generation of HCO3- for secretory fluids, as in

ocular and cerebrospinal fluids; 3) pH regulation by production of H+ from water and

CO2, as in renal acidification of urine and gastric acid secretion; and 4) facilitation of

diffusion of CO2 across membranes as in the lens of the eye [34]. The number of CA

isoforms results in functional redundancy such that organisms may survive even when a

mutation renders one isoform dysfunctional. This has been the case in humans when

deleterious mutations that occur in HCA I do not change physiological fitness because

HCA II compensates.

HCA II is well known for its presence in red cells where it efficiently stores and

converts CO2 waste in respiration as well as its maintenance of secretory fluids in the

eye. HCA II deficiency syndrome results in a variety of symptoms including renal tubular

acidosis, cerebral calcification, and osteopetrosis when inherited as an autosomal

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recessive trait [35]. Recently the over-expression of HCA IX has been associated with

several malignant tumor types; CA acidification implicated in tumor growth and

metastasis; and proposed as a possible cancer marker [36]. A drive to develop a new

class of inhibitors and markers for the enzyme may benefit from the identification of the

active site features important for proton transfer or isozyme specificity.

24

A

B Figure 1-1. The structure of HCA II. A) cartoon representation of the over all globular

structure of the alpha class Hunam CAII. The active site residues are shown as sticks Orange for the hydrophobic CO2 binding pocket and blue for the hydrophilic resudues; the zinc ion and the oxygen molecule of waters are shown as gray and red spheres, respectively. B) Close up ball-and-stick diagram of coordinating active site residues as labeled; the zinc ion and the oxygen molecule of waters are shown as gray and red spheres, respectively. The water network of the active-site is labeled W1, W2, etc. Presumed hydrogen bonds are represented as dashed red lines. The dual conformation of His64 side chain is shown in both inward and outward conformations[19].

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CHAPTER 2 KINETICS OF SPEEDING UP PROTON TRANSFER IN HUMAN CARBONIC

ANHYDRASE II

The previous chapter introduces the mechanism by which human carbonic

anhydrase II (HCA II) interconverts carbon dioxide (CO2) and bicarbonate (HCO3-).

This chapter will discuss the second stage of the reaction, the regeneration of zinc-

bound hydroxide by proton transfer. Specifically, the roles of active site hydrophilic

residues Tyr7 and Asn62 and Asn67 in tuning the pKa of the proton acceptor, His64 and

the effect on proton transfer efficiency.

As was discussed previously, the reaction catalyzed by HCA II is a two-step cycle.

The first step, in the hydration direction, is the hydration of CO2 to form bicarbonate at

the zinc. The bicarbonate becomes displaced when a water molecule diffuses into the

active site. The zinc-bound water is catalytically inactive, so a proton must be

transferred off of this water molecule to regenerate a zinc-bound hydroxide, a step that

is rate limiting for the overall catalytic cycle. This is accomplished through an ordered

network of solvent molecules that form a hydrogen-bonded wire to connect from the

zinc bound solvent to the proton shuttle residue His64 (Figure 1-1). The water network

is maintained in part by interactions with hydrophilic residues Tyr7 and Asn62 and

Asn67 of the extended active site. These residues located on either side of the proton

shuttle reside His64 (Figure 1-1), can tune the proton transfer efficiency of His64 in

equation 2 directly as well as though hydrogen boding with the water network.

Extended Active Site Hydrophilic Residues

Although the effect of the replacement of residues lining the active site cavity on

catalysis is complex, the kinetic data may indicate influences conducive to increased

proton transfer rates. Additionally Tyr7, Asn62, and Asn67 have shown little or no effect

27

on the interconversion of CO2 and bicarbonate that occurs 7-10 Å away from the zinc

[23]. It is notable that Try7 is invariant in α class CA II from a wide range of species from

chicken, rodents, bovine and the human isozymes [2]. Asn62 and Asn67 are not as

conserved in isozyme of HCA but are consistently hydrophilic except in the case of HCA

I and HCA IV (containing Val62 and Met67 respectively). Asn62 and Asn67 vary

particularly in conjunction with histidine substitutions at residue 64, such as HCA III with

Lys64 and Arg67, and HCA V with Tyr64, Thr62, and Gln67[23]. Interestingly in human

isozymes position 67 is more often a glutamine (HCA VI, VII, VIII, IX, XIV) than

asparagine (HCA II, XIII).

Hydrophobic substitutions done in these hydrophilic residues resulted in

interesting kinetics that will guide the future substitutions at these residues [23, 32].

Interest in this region of the protein is based on the mutant Y7F HCA II which showed

that the proton transfer component of catalytic dehydration was enhanced as much as

9-fold compared to wild type [23]. This increased rate of proton transfer occurred with

changes in the pKa of donor and acceptor as well as structural differences. Similar

hydrophobic mutations at position Asn62 also showed the changes in the pKa of the

donor and acceptor groups but with fewer structural changes [32].

Faster Rates of Proton Transfer

Here we discuss a number of substitutions at position 7, 62, and 67 in HCA II that

affect rate of the catalysis. Catalysis by each of the variants was studied by 18O

exchange between CO2 and water using membrane inlet mass spectrometry. We have

found that substitution at Tyr7 had no effect on the first stage of catalysis (eq 1), but

substitutions at position 62 and 67 showed some decrease in efficiency compared with

wild type. Mutations at Tyr7, Asn62, and Asn67 are shown to affect the pKa of His64.

28

These electrostatic changes alter the rate of proton transfer according to Marcus

theory.These studies emphasize the role of Tyr7 and Asn67 on long range,

intramolecular proton transfer.

Methods

Expression and Purification of Enzymes

Several variants of HCA II were created with the Stratagene Quick Change II site-

directed mutagenesis kit (La Jolla, CA) on the expression vector coding the full-length

wild type HCA II [37]. Tyr7 was replaced with Ala, Ile, Trp, Asp, Asn, Ser, and Arg.

Conservative mutations were also examined by replacing Asn62 and Asn67 with Gln

and the double substitution Tyr7 to Phe with Asn67 to Gln. The DNA sequences of each

mutant were confirmed for the entire region coding of CA in the vector. The verified

plasmids were then transformed for expression into Escherichia coli BL21(DE3)pLysS

cells from Stratagene. The transformed cells were grown in LB media containing 1.0

mM ZnSO4 and induced with IPTG to a final concentration of 1.0 mM at an OD600 of 0.6

AU [32]. Each variant was purified by affinity chromatography using p-(aminomethyl)

benzenesulfonamide coupled to agarose beads (Sigma) [38]. Protein concentrations of

variants were determined by titration with the tight-binding inhibitor ethoxzolamide and

detecting activity by 18O exchange between CO2 and water.

Kinetics 18O Exchange

Catalysis was measured by the 18O exchange method based on the measurement

by membrane inlet mass spectrometry of the depletion of 18O from species of CO2 [39].

The apparatus uses a membrane probe of silastic tubing, which is permeable to

dissolved gases and is submerged in the reaction solution. The probe is connected to

29

glass tubing that runs through a dry-ice and acetone water trap and continues to the

mass spectrometer (Extrel EXM-200) [40]. In the first of two independent stages of

catalysis, the dehydration of labeled bicarbonate has a probability of transiently labeling

the active site with 18O (eq 3). In a second stage, the protonation of the zinc-bound, 18O-

labeled hydroxide results in the release of H218O to the solvent (eq 4).

HCOO18O⎯ + EZnH2O ⇋ EZnHCOO18O⎯ ⇋ COO + EZn18OH⎯ (3) - H2O

H + His64-EZn18OH⎯ ⇋ His64-EZnH218O ⇋ His64-EZnH2O + H2

18O (4)

Two rates for the 18O exchange catalyzed by CA are obtained by this method. The

first is R1, the rate of exchange of CO2 and HCO3⎯ at chemical equilibrium, as shown in

eq 5. Here kcatexch is a rate constant for maximal interconversion of substrate and

product, KeffS is an apparent binding constant for substrate to enzyme, and S indicates

substrate, either CO2 or bicarbonate. The ratio kcatexch/Keff

CO2 is, in theory and in

practice, equal to kcat/Km for hydration obtained by steady-state methods [12].

R1/[E] = kcatexch [CO2]/( Keff

CO2 + [CO2] ) (5)

The second rate determined by this method is RH2O, which is the rate of release of

18O labeled water from the active site (eq 4). RH2O is a measure that is dependent upon

the donation of protons to the 18O-labeled zinc-bound hydroxide. In eq 6, kB is the rate

constant for proton transfer to the zinc-bound hydroxide, and (Ka)His64 and (Ka)ZnH2O are

the ionization constants of the proton donor and zinc-bound water molecule.

RH2O/[E] = kB / ([1 + (Ka)His64 /[H+]][1 + [H+]/(Ka)ZnH2O]) + RH2O (6)

Here RH2O is a pH independent contribution introduced to provide a fit to data

showing a plateau in kBobs at pH > 8. Fits of eqs 5 and 6 to the data were carried out

using Enzfitter (Biosoft).

30

The catalyzed and uncatalyzed exchanges of 18O between CO2 and water at

chemical equilibrium were measured in the absence of buffer at a total substrate

concentration of 25 mM of all species of CO2 using membrane-inlet mass spectrometry.

The total ionic strength of solution was kept at a minimum of 0.2 M by the addition of

Na2SO4. The kinetics on Y7F HCA II was reported earlier [23].

Esterase Activity

The catalysis by HCA II and mutants of the hydrolysis of 4-nitrophenylacetate was

measured by the method of Verpoorte et al. [41]. The hydrolysis was followed at 348

nm, the isosbestic point of nitrophenol and the conjugate nitrophenylate ion using the

molar absorbtivity 5.0 × 103 M-1 cm-1. A Beckman Coulter DU 800 spectrophotometer

was used to measure both the uncatalysed and carbonic anhydrase-catalysed initial

velocities. A range of buffers from pH 5.0 to 9.0 were used at 100 mM (MES, ACES,

MOPS, HEPES, TAPS, CHES, CAPS). The kinetic constants and ionization constants

were determined from the pH profiles of kcat/KM by nonlinear least-squares methods to a

single ionization with a maximum at high pH (Enzfitter, Elsevier-Biosoft, Cambridge,

U.K.).The rate constants kenz reported here represent kcat/Km for the catalyzed

hydrolysis. The value of KM is too large to measure kcat.

Results

The site-specific mutants of HCA II in Table 2-1 were investigated for their catalytic

properties using membrane inlet mass spectrometry measuring the rate of exchange of

18O between CO2 and water. The efficiency of the catalyzed hydration of CO2 was

measured as the rate constant kcatexch/Keff

CO2 (eq 5) over a pH range of 5.0 to 9.0. The

pH profiles for kcatexch/Keff

CO2 by the Tyr7 mutants under study were adequately fit to a

single ionization and appeared similar to that of the wild type (Figure 2-1, 2-2). The

31

Asn62 and Asn67 mutations shown in Figure 2-3 some deviations compared to the wild

type. The resulting maximal values of kcatexch/Keff

CO2 (Table 2-1) represent catalytic

activity in the hydration direction and were essentially identical to that of wild type

except for N62Q and N67Q that showed a slight decrease in efficiency. This lower

kcatexch/Keff

CO2 was similar to those observed during the substitution of hydrophobic

residues at position 62 [32]. The variation was greater for the activities kcat/Km for the

catalyzed hydrolysis of p-nitrophenylacetate (Table 2-1) but followed the trend of the

maximal values of kcatexch/Keff

CO2. This probably reflects the larger size of the substrate

for the ester hydrolysis which is influenced more by amino acid changes near the mouth

of the active site cavity. The resulting values of pKa ZnH2O representing the ionization of

the zinc-bound water for each mutant are listed in Table 2-2. The data demonstrate that

the values of pKaZnH2O of the zinc-bound water are near 7 for wild type and mutants.

These pKa ZnH2O values were determined both by measurement of esterase activity and

18O exchange (Table 2-2).

The pH profiles of the rate constant RH2O/[E] (Figures 2-4, 2-5, and 2-6) provide

three constants relevant to the catalysis, according to eq 6. The first is an estimate of

pKa ZnH2O, values which are given in Table 2-2 and generally are consistent with the

values described in the above paragraph. Exceptions are Y7A, Y7S, Y7R and

Y7F+N67Q (Table 2-2; Figure 2-2). These differences may be due to different

properties of the active site for the processes of eq 3 and eq 4, or possibly difficulty

interpreting the irregular curves for RH2O during catalysis by Y7A and Y7S (Figure 2-2).

The RH2O/[E] profile of Y7D did not have sufficient bell shape to assign the pKa values.

In later construction of a free energy plot, we have used data only from RH2O.

32

The second constant is a value of pKa His64 (Table 2-2). These values are uniformly

lower for replacements at residue 7, 62, and 67 than the value of pKa His64 at 7.2 in wild

type HCA II (Table 2-2). Such a shift in pKa His64 is associated with the inward

coordination of the side chain of His 64 [23, 32] and its more hydrophobic environment

than in wild type HCA II in which this side chain appears about equally in inward and

outward orientations[19, 24]. The third constant is the maximal, pH-independent value

of kB describing intramolecular proton transfer in the dehydration direction obtained by a

fit to eq 6 to the pH profiles for RH2O/[E] (Table 2-2; Figure 2-4, 2-5, and 2-6). The pH

profiles for RH2O, from which kB values were obtained, were more difficult to fit for Y7D

and Y7N than the bell curve of wild type HCA II and other variants (Figure 2-5). All of

the variants at position 7, 62, and 67 showed rates of proton transfer equally to (Y7A,

Y7D) or greater than the wild type enzyme (Y7R, N62Q, Y7S, N67Q, Y7W, Y7I, Y7N,

Y7F, and Y7F+N67Q in order of increasing kB ). The data for Y7I were consistent with a

value of kB of 2.3 μs-1. The greatest changes in kB among these variants were Y7F with

a value of 7 μs-1 and Y7F+N67Q with a value of 12.5 μs-1 (Table 2-1).

Discussion

A notable result of this study of variants at residue 7 of HCA II is that the rate

constants kcatexch/Keff

CO2 for the first stage of catalysis were unchanged. The

substitutions of Tyr7 did not affect the ability of the protein to carry out the first stage of

the catalysis. This appears to be a result of the amino acid substitutions being at least

7Å away from the zinc where the interconversion between CO2 and bicarbonate occurs.

The substitutions N62Q and N67Q caused substantial change in the rate constants

kcatexch/Keff

CO2 which is not explained by values of the pKa of the zinc-bound water.

However, none of the variants in Table 2-1, had a kB for proton transfer lower than that

33

of the wild type. This supports a role for Tyr7, Asn62, and Asn67 in fine tuning the rate

of intramolecular proton transfer.

The changes in kB were examined in relationship to the donor and acceptor pKa

values on a free energy plot in Figure 2-3. The open circles are rate constants for proton

transfer during catalysis by H64A HCA II determined by enhancement of catalysis when

proton donors are exogenous derivatives of imidazole and pyridine [16]. These data are

fit by Marcus theory applied to proton transfer [42, 43], represented by the solid line of

Figure 2-3. We assume this line represents the dependence of kB on ΔpKa within the

active site of HCA II. The significance of this fit is that these values of kB are for proton

transfer from donors not attached to the enzyme through chemical bonds, free of many

restraints. Interestingly, the values of kB for wild type HCA II and nearly all of the

variants of Table 2-1, except Y7F, and Y7F+N67Q HCA II, fall on or near this Marcus

line (Figure 2-7). We can conclude that the small variation in kB for the majority of these

mutants were possibly due to small changes in the electrostatics of the active site

thourgh possible changes in His64 or the water network that altered its pka.

The large increase in the kB for the Y7F, and Y7F+N67Q HCA II mutants was not

explained simply by Marcus theory but will be discussed with respect to structural

changes in Conclusions (Chapter 5). A double mutant catalytic cycle showed the

change in proton transfer rates between the single and double mutants from wild type

HCA II. This compares the changes in free energy barriers ΔG according to ΔG =-

RTln[kB (mutant2)/kB (mutant1). The large negative ΔG values in the cycle of these

mutants indicate that the increases in activity compared to wild type result in significant

decreases in the free energy barriers of proton transfer. The change in the activation

34

barrier for the double mutant ΔG1+2 in Figure 2-8 for Y7F+N67Q HCA II (-1360 cal/mol)

was greater than sum of the individual mutation Y7F and N67Q HCA II (-820 cal/mol).

Thus we classify this double mutant interaction as synergistic (greater than the sum of

the individual changes) and results in an increase in the proton shuttle efficiency of

His64.

Kinetic changes we observed maybe better understood in the light of structural

changes in the active site water network as well as the conformation of His64 in these

mutations and variation of other key residues (See Conclusions). The results help

confirm a major driving force of proton transfer is the electrostatics of the active site that

defines the pKa of both the zinc-bound solvent and His64. These changes in pKa can

directly affect the change in proton transfer consistent with Marcus theory. The ability to

increase the rate of proton transfer by nearly 15 fold in Y7F+N67Q HCA II has never

been seen before.

35

Table 2-1. Kinetic rate constants for HCA II and mutants. The maximal values of the rate constants for hydration of CO2, hydrolysis of 4-nitrophenylacetate, and proton transfer catalyzed by HCA II and variants. Rate constants were derived from the kinetic curves for each substitution by a fit of the data. a Measured from the exchange of 18O between CO2 and water using eq 5 in the hydration direction. b Measured from the fit of the rate constants for ester hydrolysis to a single ionization. c Measured from the exchange of 18O between CO2 and water using eq 6 in the dehydration direction. d Data are from Fisher et al. [23]. e These are maximal values of RH2O/[E] since incomplete pH profiles (Figure 2-2) did not allow an adequate determination of kB by a fit of eq 6.

Enzyme kcat

exch/KeffCO2

CO2 hydration (μM-1s-1) a

kcat/Km esterase (M-1s-1) b

kB proton transfer (μs-1) c

Wild Type 120± 2 2800± 100 0.8± 0.1 Y7I 130± 3 2400± 100 2.3± 0.2 Y7A 140± 5 2300± 100 0.8± 0.3 Y7W 140± 9 1700± 10 1.8± 0.1 Y7F d 120± 2 4400± 10 7.0± 0.2 Y7D 130± 8 1800± 80 0.8 e± 0.1 Y7N 120± 3 1200± 60 2.5 e± 0.1 Y7S 120± 4 1700± 50 1.5± 0.1 Y7R 120± 2 1600± 60 1.0± 0.1 N62Q 50± 8 2800± 80 1.0± 0.4 N67Q 50± 8 5600± 100 1.7± 0.3 Y7F+N67Q 100± 2 7500± 150 12.5± 2

36

Table 2-2. Values of apparent pKa obtained by various kinetic measurements of

catalysis in HCA II and mutants. a The pKa was derived from the fits of eq 6. b The pKa was determined from a fit of eq 5. The observed pKa values from a second ionization by some mutants; were not included in this table. c These data from Fisher et al.[23]. d The Y7D HCA II data for RH2O/[E] did not have sufficient bell-shape to be adequately fit by eq 6 (Figure 2-2).

Enzyme pKa His64a

(eq 6) pKa ZnH2O a

(eq 6) pKa ZnH2O b

(kcatexch/Keff

CO2) pKa ZnH2O (esterase)

Wild Type 7.2±0.1 6.8±0.1 6.9± 0.1 6.9± 0.1 Y7I 6.2±0.1 6.8±0.1 7.1± 0.1 6.9± 0.1 Y7A 6.4±0.2 6.4±0.3 7.0± 0.1 7.2± 0.1 Y7W 6.9±0.2 7.0±0.3 7.2 ± 0.1 7.0± 0.1 Y7F e 6.0 c ±0.2 7.0c±0.2 7.1c± 0.3 7.0± 0.1 Y7D --d -- d 6.4± 0.1 6.6± 0.2 Y7N 6.2±0.1 6.8±0.1 6.8± 0.1 6.4± 0.1 Y7S 6.2±0.1 6.2±0.3 7.4± 0.1 7.1± 0.1 Y7R 6.4±0.1 6.4±0.1 7.0± 0.1 6.9± 0.1 N62Q 6.6±0.1 7.3±0.1 6.7± 0.1 7.9± 0.3 N67Q 6.6±0.1 6.7±0.1 6.5± 0.1 7.1± 0.1 Y7F+N67Q 6.3±0.1 6.3±0.1 7.0± 0.1 7.5± 0.2

37

Figure 2-1. The pH profiles for kcat

ex/KeffCO2 (M-1s-1) for the hydration of CO2 catalyzed

by HCA II and hydrophobic Tyr7 substitutions. The log of hydration of CO2 relative to the amount of enzyme is described from pH 5.0 to 9.0 for: Y7A HCA II (◊), Y7I HCA II (□), Y7W HCA II (○), Y7F HCA II (Δ), and wild type (♦). Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 and sufficient Na2SO4 to maintain 0.2 M ionic strength.

38

Figure 2-2. The pH profiles for kcat

ex/KeffCO2 (M-1s-1) for the hydration of CO2 catalyzed

by HCA II and polar substitutions at Tyr7. The log of hydration of CO2 relative to the amount of enzyme is described from pH 5.0 to 9.0 for: (♦)Y7D HCA II (Δ), Y7N HCA II (■), Y7R HCA II (□), Y7S HCA II (○), and wild type HCA II (♦). Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 and sufficient Na2SO4 to maintain 0.2 M ionic strength.

39

Figure 2-3. The pH profiles for kcat

ex/KeffCO2 (M-1s-1) for the hydration of CO2 catalyzed

by HCA II and conservative substitutions at Tyr7, Asn62 and Asn67. The log of hydration of CO2 relative to the amount of enzyme is described from pH 5.0 to 9.0 for: N62Q HCA II (□);N67Q HCA II (○);Y7F HCA II (Δ); Y7F+N67Q HCA II (■); and wild-type HCA II (♦). Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 and sufficient Na2SO4 to maintain 0.2 M ionic strength.

40

Figure 2-4. The pH profiles for the proton-transfer dependent rate of release of 18O-

labeled water by HCA II and hydrophobic substitutions. Kinetic profiles were determined by membrane inlet mass spectrometry for: Y7A HCA II (◊), Y7I HCA II (□), Y7W HCA II (○), Y7F HCA II (Δ), and wild type (♦). Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 at sufficient Na2SO4 to maintain 0.2 M ionic strength. No buffers were added.

41

Figure 2-5. The pH profiles for the proton-transfer dependent rate of release of 18O-

labeled water by HCA II polar substitutions at Tyr7. Kinetic profiles were determined by membrane inlet mass spectrometry for: (♦)Y7D HCA II (Δ), Y7N HCA II (■), Y7R HCA II (□), Y7S HCA II (○), and wild type HCA II (♦) . Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 at sufficient Na2SO4 to maintain 0.2 M ionic strength. No buffers were added.

42

Figure 2-6. The pH profiles for the proton-transfer dependent rate of release of 18O-

labeled water by HCA II and conservative substitutions at Tyr7, Asn62 and Asn67. The kinetic profiles were determined by membrane inlet mass spectrometry for: N62Q HCA II (□);N67Q HCA II (○);Y7F HCA II (Δ); Y7F+N67Q HCA II (■); and wild-type HCA II (♦).Data were obtained by 18O exchange between CO2 and water using solutions containing 25 mM of all species of CO2 at sufficient Na2SO4 to maintain 0.2 M ionic strength. No buffers were added.

43

Figure 2-7. Free energy plot of proton transfer in HCA II and variants. The logarithm of the rate constant for proton transfer kB (s-1) versus ΔpKa (from pKa ZnH2O – pKa

His64) determined from the RH2O/[E] pH profiles for the wild type and the mutants of HCA II identified on the plot (◊ 25°C data, □ 10°C data); and H64A HCA II (●) from An et al. [44] with proton transfer provided predominantly by derivatives of imidazole and pyridine acting as exogenous proton donors with the solid line a best fit of Marcus Rate Theory [42].

44

45

Figure 2-8. Double mutant catalytic cycle. Comparisons of the rate constants for

intramolecular proton transfer for HCA II and mutants. The values of kB (ms-1) appear beneath each designated mutant, and the values adjacent to the arrows are the free energy changes (ΔG in kcal/mol) for the barriers to proton transfer for the corresponding mutations calculated from kB (ΔG =-RTln[kB (mutant2)/kB (mutant1)]. Data were obtained by 18O exchange between CO2 and water using solutions at 10 °C containing 25 mM of all species of CO2 at sufficient Na2SO4 to maintain 0.2 M ionic strength. No buffers were added.

CHAPTER 3 STRUCTURE OF PROTON TRANSFER PATHWAYS IN HUMAN CARBONIC

ANHYDRASE II

The previous chapter identified changes in proton transfer rates for mutants of

HCA II in the region of His64.This chapter will describe structural changes in the active

site due to these same mutations in HCA II. The X-ray crystal structures allow us to

examine the active site water network, conformation of His64 and changes in active site

residues. The discussion will examine the kinetic changes observed in several of the

mutants in Chapter 2 with the crystal structures of Y7I, N62Q, N67Q, and Y7F+N67Q

HCA II reported here.

Structural Role of Water Network in Proton Transfer

The active site cavity of HCA II has distinct hydrophilic and hydrophobic surfaces

with a zinc atom at the bottom of a conical cavity. An ordered chain of hydrogen

bonded, water molecules (W1, W2, W3a and W3b) extends from the zinc-bound water

to the proton shuttle residue His64 (Figure 1-1). The water network is maintained in part

by interactions with hydrophilic residues within the active site cavity (Tyr7, Asn62,

Asn67, Thr200). The significance of this ordered water network in rapid transfer of

protons has been studied in various mutants of HCA II [9, 19, 23, 32] as well as

molecular dynamic simulations[28]. Computational studies indicate a more rapid proton

transfer through a single, non-branched chain of water molecules compared with

branched chains [28, 45, 46].

Proton Shuttle Residue

The final step in the transport of protons out of the active site is the proton shuttle

residue His64, which is believed to bridge the active site with the bulk solvent. Crystal

structures of HCA II show that the side chain of His64 exists in two conformations

46

referred to as the inward and outward conformations [19, 24]. They also observed a pH

dependence of the His64 residue orientation where structures at pH 8.0 to 9.0 had

mostly inward His64 and at pH 5.7 had mostly outward His64 in wild type HCA II. The

rotation of His64 from inward to outward has been limited by steric interactions with

Trp5 and Asn62 such that a rotation about χ1 appears limited to 100° [47]. The

implications of the inward and outward orientations of the proton shuttle for facile proton

transfer in CA are still under investigation.

Overall HCA II is a small and compact protein with the possible exception of the N-

terminal region (residues 1 to 24) that is more loosely connected to the rest of the

molecule. N-terminal residues 1-3 are usually disordered in the crystal structures. A

cluster of aromatic residues at the N-terminus of HCA II consisting of Trp5, Tyr7, Trp16,

and Phe20 has been suggested to assist in the anchoring of this region to the rest of the

enzyme [18]. It has also been shown that the removal of up to 24 residues from the N-

terminal region does not result in a major loss of protein stability or enzyme activity. The

active site folds first and independently of the N-terminal region.

Methods

X-Ray Crystallography

Crystals of the mutants Y7I, N62Q, N67Q and Y7F+N67Q HCA II were obtained

using the hanging drop method [48]. The crystallization drops were prepared by mixing

5 μL of protein [concentration of ~15 mg/mL in 10 mM Tris-HCl (pH 8.0)] with 5 μL of the

precipitant solution. The precipitants were mutant specific: 2.8 M ammonium sulfate with

50 mM Tris-Cl (pH 8.5) for Y7I, 1.25 M sodium citrate with 100 mM Tris-Cl (pH 8.0) for

N62Q and 2.6 M ammonium sulfate, 5.0 mM HgCl, and 100 mM Tris-Cl (pH 8.0) for

N67Q]. The drops were equilibrated at room temperature against a well of 1000 μL

47

precipitant solution. Crystals were observed within a week of the crystallization setup at

293K.

The x-ray diffraction of Y7I HCA II was obtained at room temperature, using an R-

AXIS IV++ image plate system with Osmic mirrors and a Rigaku HU-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 7 min exposure per image. The N67Q, N62Q,

and Y7F+N67Q HCA II crystals underwent a quick cryo cooling with a 30% glycerol

cryo-protection before mounting for diffraction. The x-ray diffraction of these crystals

was obtained at 100K, with a liquid nitrogen stream, using an R-AXIS V++ optic system

from Viarimax HR a Rigaku HU-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°. Data set statistics for all three crystals are given in

Table 3-1.

Diffraction data were indexed, integrated, and scaled using the HKL2000 program

package [49]. The model building was done manually with the program Coot [50], and

refinement was carried out with Phenix suite. The data sets were phased to the 1.54 Å

resolution structure of wild-type HCA II (PDB ID: 1tbt) with the waters removed and both

His64 and the appropriate Asn 62 or 67 residues mutated to Ala to prevent any

introduction of bias. The initial maps were used to confirm and fit the appropriate side

chain conformations of His64 and other mutated residues. Waters were then placed and

confirmed in Coot during several rounds of refinement until the Rcryst reached

convergence. Final refinement statistics for all four variants are given in Table 3-1.

48

These structures are compared to the 2ILI (1.05 Å resolution wt HCA II structure) and

the 2NXT (1.15 Å resolution Y7F HCA II structure).

Thermodynamic Stability

Differential scanning calorimetry (DSC) experiments were performed using a VP-

DSC calorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of ~0.5 ml. Tyr7

variants of HCA II were buffered in 50 mM Tris-HCl, pH 8.0 at protein concentrations of

50 μM. DSC scans were collected from 30 to 90 °C with a scan rate of 60 °C/hour. The

calorimetric enthalpies (ΔH ºm) of unfolding were calculated by integrating the area

under the peaks in the thermograms after adjusting the pre- and post-transition

baselines. Heat capacity (ΔCp) of protein unfolding obtained by plotting calorimetric

enthalphy (ΔH ºm) vs melting temperature (Tm).

Results

The crystal structure of the four variants N62Q, N67Q, Y7F+N67Q and Y7I were

solved to resolution 1.5-1.6 Å (Table 3-1).

Structure of Y7I HCA II

The Y7I HCA II crystal was well ordered, and diffracted X-rays to 1.5 Å

resolution. The structure of Y7I HCA II was similar to wild type in that the residues

coordinating the zinc, and those in the active site cavity (with the exception of residue

seven) are unchanged (Figure 3-1). His64 showed a predominantly inward orientation

similar to Y7F HCA II reported earlier [23]. However, the structure of Y7I HCA II showed

changes in the N-terminus that have not been observed in other Tyr7 mutants. The

altered location of His10 in this structure replaces Trp5 in the wild types pie stacking

interaction in wild type with His64 which may help stabilize the inward conformation of

His64 in this mutant. Most notably there was a novel conformation of residues 4 to 11 of

49

the N-terminal chain (Figure 3-2). Several new interactions are observed here for

residues 4,6,8-11 that are different from those in the wild type which may help stabilize

this new N-terminal conformation. The amino-terminal (N-terminal) residues 1 to 3 were

not observed in the crystal structure presumably because of disorder. This N-terminal

conformation in Y7I HCA II caused the side chain of Ile7 to point away from the metal in

the active site; the side chain of Ile7 does not occupy a position analogous to the side

chain of Tyr7 in wild type, which lies between Thr200 and His64 (Figure 3-2).

Despite the differences in structure of the N-terminal residues of Y7I and wild

type, the geometry of the residues coordinating the zinc and the active site solvent

structures were not altered (Figures 3-1). The observed changes in the N-terminal

residues may be due to protein stability which was examined by DSC described below.

The altered conformation in Y7I was not expected because the Y7F structure had an N-

terminal fold similar to wild type. The N-terminal fold in wild type and Y7F position side

chain of residue seven toward the active site near W3a. The residue at position seven is

held by week van der waals forces of the surrounding amino acids side chains and the

hydrogen bond to W3a but no other direst interactions are observed. The structure also

showed that Ile7 of Y7I interacted with other residues in one of the crystal contact

regions. Its amide nitrogen forms a hydrogen bond with the carboxyl side chain of Glu

238 and its side chain is in hydrophobic contact with Leu 240 (Figure 3-3). These

interacting amino-acids are located in a single crystal-contact patch. It is possible that

the altered orientation of the N-terminus of Y7I HCA II is a consequence of crystal

contacts. It is interesting to note however, that this mutant crystallizes in the same

space group as the wild type enzyme.

50

Role of Tyr7 in Thermal Stability of HCA II

The thermal stability of HCA II mutants was determined by differential scanning

calorimetry (DSC). A single peak representing the main unfolding transition was

observed for Y7A, Y7W, Y7D, Y7N, Y7S,and Y7R HCA II as well as wild type, Y7I and

Y7F HCA II as shown in Figure 3-4. The main unfolding transition of the wild-type

enzyme Tm occurred at 59.5 ± 0.5 °C and that of Y7I occurred at 51.8 ± 0.5 °C. The

values of Tm for each of the remaining variants of were between 49.3 and 52.5 °C

except Y7F which had a distinctly broad transition with a Tm of 55.8 ± 0.5 °C (Table 3-2).

Structures of N62Q, N67Q and Y7F+N67Q HCA II

The crystal structure of the three variants N62Q, N67Q, Y7F+N67Q of HCA II were

solved to 1.5-1.6 Å resolution and completeness greater than 90% (Figure 3-5). The

final statistics of the structure including the Rcrysts of 17% and Rfrees 20% are listed in

Table 3-1. Overall, no major structural perturbations were observed with Cα rmsd less

than 0.27 Å for all three variants when compared to the wild-type HCA II (2ili)[47].

The structures of N62Q, N67Q, Y7F+N67Q HCA II were similar to wild type in

that the residues coordinating the zinc, and those in the active site cavity other than the

mutated residue are unchanged (Figure 3-5). The proton shuttle residue, His64 has

been shown to occupy two conformations in wild-type HCA II. The crystal structures of

the mutants determined here showed only one orientation of the side chain. The

outward confirmation was dominant in both the N62Q and N67Q while the inward

conformation was observed in the Y7F+N67Q. In N67Q and N67Q the distance from the

zinc to the proton shuttle residue was greater by about 0.5 Å more than wild type in its

out conformation (Table 3-3). In the case of the Y7F+N67Q we do not see any density

in the outward conformation and a hydrogen bond of 2.8 Å occurs with W2 (Table 3-3).

51

The hydrogen bonded solvent network in the active site cavity was altered to

various extents in these mutants (Table 3-2). The N62Q structure showed additional

waters in the region of W2 which have lower occupancy (0.3) and may be other

conformations of W2 or a result of H64 being in the out conformation (Figure 3-5A).

N67Q had a water network most similar to wild type and despite having a predominantly

outward His64 orientation did not show an extra water molecule. The Y7F+N67Q

mutant had a less branched water as W3a was more than 3.5 Å away from W2. This

makes the network in Y7F+N67Q more similar to Y7F HCA II with His64 being within

hydrogen bonding distance to W2.

Discussion

The goal of this work was to elucidate the role in catalysis of residues 7, 62, and

67 on the hydrophilic side of the active site cavity in HCA II, in particular its influence on

the structure of ordered solvent, and on the properties of the proton shuttle His64.

Despite the large changes in the N-terminal region of Y7I HCA II, the structure of

the active site is remarkably similar to that of the wild type enzyme. This includes the

network of ordered water molecules (Figures 3-1,3-2), unlike Y7F HCA II in which water

molecule W3a is not observed. In the crystal lattice, Ile7 rests in one of the crystal

contact regions. Its amide nitrogen forms a hydrogen bond with the carboxyl side chain

of Glu 238 and its side chain is in hydrophobic contact with Leu 240 (Figure 3-4). These

interacting amino-acids are present in a single crystal-contact patch. It is possible that

the altered orientation of the N-terminus of Y7I HCA II is a consequence of crystal

contacts. It is interesting to note however, that this mutant crystallizes in the same

space group as the wild type enzyme. After repeated attempts to crystallize other Tyr7

mutants listed in Table 2-1, only Y7I, Y7F, and Y7F+N67Q HCA II have been

52

successful. It is possible that mutations at Try 7 produce a disordered (or alternate) N-

terminal conformation giving rise to structural heterogeneity in the samples, which is

likely to deter crystal growth. Truncation of as many as 24 residues of the N-terminal

end of HCA II does not prevent the remaining protein from folding correctly, and the

structure of the N-terminus has been shown to form very late in folding [51].

A comparison of the characteristics of the three structures with Tyr7 mutations

reveals that Y7I, Y7F, and Y7F+N67Q HCA II all had His64 in the inward orientation.

However, they differ in their values of kB, almost 3 and 7, and 15 fold greater than wild

type respectively (Table 2-1). The Y7I structure was different from both the other Tyr7

mutants in that the water network was nearly the same as in the wild type possibly due

to the altered N-terminus. Y7I HCA II also showed increased distance between the zinc

and His64 (7.9 Ǻ) as well as between W2 and His64 (3.7 Ǻ) relative to Y7F, Y7F+N67Q,

and wild type HCA II. These structural changes may increase the energetic barrier to

proton transfer and explain why the increase in ΔpKa over wild type followed the

theoretical Marcus in Y7I HCA II (Table 3-4).

Interestingly, the values of kB for wild type HCA II and nearly all of the variants of

Table 2-1, except Y7F, and Y7F+N67Q HCA II fall on or near this Marcus line (Figure 2-

7). These significantly increased kB values were not simply explained the changes

observed in ΔpKa by the application of Marucs theory as described in Chapter 2. The

structural features in contrast to Y7I (which followed Marcus theory) were decreased

distance between His64 and the zinc bound solvent, and altered water networks. The

loss of W3a in 7F or the 4.3 Ǻ distance preventing hydrogen bonding between W2 and

W3a in Y7F+N67Q decrease the branching at W2 and showed a possible single strand

53

of hydrogen bonding waters between the zinc and His64. The Y7F+N67Q structure

showed a significantly shorter distance (2.8 Ǻ) from W2 to His64 than in Y7F and wild

type HCA II (3.2 Ǻ) structures.

It is very difficult to isolate the effect of the conformation of His64 on the rate of

proton transfer. The N67Q and N62Q HCA II both showed outward conformations

compared to the Try7 mutants. Despite the outward orientation of His64 these enzymes

still show proton transfer rates faster than wild type. The increase in kB for N67Q follows

the theoretical Marcus curve for HCA II based mainly on the change in ΔpKa. The N62Q

mutant on the other hand falls slightly below the Marcus theory line which may be due

to the additional water filling the additional space due to the outward conformation of

His64. It is interesting that we do not see additional water in the N67Q structure to fill

this space and this may indicate why the proton transfer rate followed Marcus theory

more closely than the N62Q mutant. Previous substitutions at Asn62 also showed

decreases in kB regardless of the conformation of His64[32].

We suggest that the reason the value of kB for Y7F and Y7F+N67Q HCA II were

considerably above the Marcus line of Figure 2-7 while the other mutants lie closer to

the line was due to structural changes in the proton transfer pathway that decrease the

energetic barrier to proton transfer. The significant difference first observed in Y7F was

a less branched, hydrogen-bonded water structure in the active site cavity (Figure 3-1).

In solution these water structures have a lifetime in the picosecond range; however, the

ordered water in the crystal structures provides a clue to the more stable water

structures in catalysis. Computational studies of the proton transfer step in catalysis by

carbonic anhydrase show that water wires consisting of fewer molecules transfer

54

protons more efficiently [52]. Wild-type HCA II has an identical water structure to Y7I

and N67Q in the active-site cavity with a branched cluster of four ordered water

molecules between His64 and the zinc bound water (Figures 3-1, 3-4). The additional

water in the active site of N62Q lengthens the water network between zinc and the

His64 and may reduce the proton transfer rate to 1.0 μs-1 (Table 3.4). In contrast, Y7F

has a smaller, unbranched cluster of three water molecules that is more stable and

provides a proton transfer pathway of lower energy barrier than wild type [52]. This was

similar to the Y7F+N67Q double mutant which had a distance between W2 and W3a

greater than that of a hydrogen bond (Table 3-2). This provides an explanation of why

Y7F and Y7F+N67Q do not lie on the line of Figure 2-7 like wild type, as well as other

mutants. The combination of more conventional hydrogen bond distances connecting

the zinc solvent to His64 though only two molecules of water compared to the branched

network in wild type HCA II may provide significantly more favorable pathway for proton

transfer.

It is interesting to speculate why Tyr7 is conserved when substitution can enhance

the rate of maximal catalysis. Calorimetry showed that the replacements of Try7 in HCA

II decreased the thermal stability of the protein by 7 – 10 °C, except for Y7F which was

decreased about 4 °C. This decrease indicates that Tyr 7 stabilizes the enzyme,

although it is unclear how it does this since the side chain of Tyr 7 has no apparent

interactions with other residues in the crystal structure. At any rate, this decreased

stabilization may be a factor to explain the occurrence of Tyr7 in many of the carbonic

anhydrases in the α class. Tyr7 also promotes folding of the N-terminal chain, which

appears to have another role. A conserved N-terminus is consistent with this region of

55

56

HCA II binding to the Cl⎯/bicarbonate anion exchange proteins (AE) [53]. Recent

deletion and mutation studies have shown the numerous histidine residues (His3,His4,

His10, His15 and His 17) found in the N-terminus of HCA II represent an acidic motif

important for binding the C-terminus of AE-1 and AE-2 [53].

Table 3-1. Dataset, refinement, and final model statistics for the crystallographic study of N62Q, N67Q HCA II, N67Q+Y7F HCA II, and Y7I HCA II. aRsym = Σ |I - <I>|/ Σ <I>. bRcryst = (Σ |Fo| - |Fc|/ Σ |Fobs| ) x 100 . cRfree is calculated in same manner as Rcryst, except that it uses 5% of the reflection data omitted from refinement. Values in parenthesis represent highest resolution bin. N62Q N67Q Y7F+N67Q Y7I space group P21 P21 P21 P21 unit cell dimensions: a b c

42.8 41.7 72.9

42.6 41.7 73.0

42.8 41.6 73.0

42.8 41.6 73.2

β (deg)] 104.1 104.3 104.5 104.9 resolution (Å) highest shell

50.0-1.50 (1.55-1.50)

50.0-1.50 (1.55-1.50)

50.0-1.60 (1.66-1.60)

20.0-1.50 (1.55-1.50)

numberofunique reflections

36184 (3410)

35820 (3408)

31729 (3143)

21266 (2142)

completeness (%) 92.5(80.5) 90.7 (80.6) 96.5 (85.8) 91.7 (92.6) redundancy 3.6 (2.5) 4.2 (3.0) 3.8 (3.6) Rsymm a 0.108 (0.286) 0.085 (0.448) 0.106 (0.339) 0.075 (0.349)numberofprotein atoms

2467 2456 2470 2059

numberof solventatoms

307 351 314 186

Rcryst b 15.6 18.5 16.5 16.6

Rfree c 16.7 21.4 20.1 21.1

average B factor (Å2): main chain 11.6 18.2 19.8 17.3 sidechain 15.5 21.4 22.0 22.7 Zn 10.4 11.5 10.5 11.7 solvent 26.8 29.6 28.7 30.2

57

Table 3-2. Thermodynamics of unfolding of Y7 variants and wild type HCA II. a

Calorimetric parameters determined by DSC. T1/2 – is the temperature at the width of half-peak height

Enzyme Tm (ºC)a ΔH ºm (kcal mol-1)a Range (ºC) a T1/2 (ºC) a

wt 59.5±0.5 240.0±2.0 48.0-70.0 3.6±0.1

Y7I 51.8±0.5 180.0±2.0 40.0-61.0 5.6±0.1

Y7A 52.5±0.5 90.0±2.0 39.0-61.0 5.6±0.1

Y7W 52.0±0.5 86.0±2.0 42.0-60.0 5.3±0.1

Y7F 55.8±0.5 55.0±2.0 32.0-76.0 9.1±0.1

Y7D 52.0±0.5 182.0±2.0 40.0-60.0 5.0±0.1

Y7N 51.8±0.5 132.0±2.0 42.0-61.0 5.3±0.1

Y7R 49.3±0.5 138.0±2.0 41.9-55.3 5.0±0.1

Y7S 50.4±0.5 155.0±2.0 40.2-54.9 6.1±0.1

58

59

Table 3-3. Comparison of proposed hydrogen bond networks for wild type, Y7F, Y7F+N67Q, N67Q, N62Q, and Y7I HCA II. The distances (Å) between oxygen atoms of water molecules and hydrophilic residues in the active site of wild type (wt) HCA II (PDB ID: 2ILI), Y7F (PDB ID: 2NXT), Y7F+N67Q, N67Q, N62Q, and Y7I HCA II crystal structures. Oz is the oxygen atom of the Zn-OH-

/H2O. The 67 and 62 distances are those represented in Figuess 1-1, 3-1, and 3-5 by the red dashed line to the respective residue. The values for both the in and out conformation of His64 are listed for wild type as in/out. Distances for W4 in N62Q HCA II are not listed.

wt Y7F Y7F+N67Q N67Q N62Q Y7I Zn - Oz 1.9 1.9 2.1 2.1 1.9 2 Oz - Oγ1 of T199 3.0 2.7 2.6 2.7 2.7 2.7 Oz -W1 2.7 2.7 2.7 2.7 2.6 2.6 W1- Oγ1 of T200 2.8 2.8 2.8 2.7 2.8 2.8 W1-W2 2.7 2.6 2.6 2.9 2.4 2.7 W2-W3a 2.8 n/a 4.3 2.9 3.8 2.8 W3a- OH of Y7 2.8 n/a n/a 2.8 2.8 n/a W2-W3b 2.7 2.6 3.1 2.4 2.6 2.8 W3b-67 3.6 3.7 2.9 2.6 2.8 2.8 W3b-62 3.0 3.1 2.8 3.6 3.3 3.1 W2- Nδ1 of H64 3.2/6.3 3.2 2.8 6.7 5.1 3.7 Oz - Nδ1 of H64 7.2/10.0 7.1 6.7 10.5 10.4 7.6 Zn – Nδ1 of H64 7.4/10.5 7.5 7.0 11 10.8 7.9

Table 3-4. Comparison of the features proposed to regulate proton transfer rates in the protein environment in HCA II and selected variants.

wt Y7F Y7F+N67Q N67Q N62Q Y7I

kB (μs-1) 1.3 3.9 12.5 1.7 1.0 2.7

ΔpKa 0.7 1.0 0.0 0.0 0.7 2.7

Waters in network

W1, W2, W3a, W3b W1, W2, W3b W1, W2, W3a,

W3b W1, W2, W3a,

W3b W1, W2, W3a,

W3b, W4 W1, W2, W3a,

W3b Zinc-Oz to Nδ

His64 (Å) 7.2 7.1 6.7 10.5 10.4 7.6

H64 conformation in/out in in out out in

60

A

B

Figure 3-1. Crystal structures of the active sites of Y7I and Y7F HCA II. A) The

structure for Y7I HCA II in yellow overlay the wild type in gray. B) The structure for Y7F HCA II (PDB ID 2NXT) [21]. A ball-and-stick diagram of coordinating active site residues as labeled; the zinc ion and the oxygen molecule of waters are shown as gray and red spheres, respectively. The water network of the active-site is labeled W1, W2, etc. Presumed hydrogen bonds are represented as dashed red lines.

61

A

B Figure 3-2. Overall comparison of crystal structures of wild-type HCA II and Y7I HCA II

A) The superimposed crystal structures of wild-type HCA II and Y7I HCA II. B) The close up of the N-terminal region of superimposed crystal structures of wild-type HCA II and Y7I HCA II. The superimposed enzyme was represented as a surface except the N-terminus region was represented as a ribbon. The N-terminus of (yellow) wild type; and (green) Y7I HCA II is represented as ribbon, while the respective amino acids at position 7 as sticks. The hydrophobic and hydrophilic regions of the active-site are rendered orange and blue respectively. The active site zinc is depicted as a grey sphere.

62

A

B

Figure 3-3. Representation of crystal contacts in Y7I HCA II. A) A cartoon and stick view through a small section of the crystal lattice. B) Zoom-in of the inset in part A depicting the residues involved in crystal contacts. Dashed red line (2.9Å), hydrogen bond; dashed green lines (≤ 3.6Å), all other contacts.

63

64

Figure 3-4. Differential scanning calorimetry profiles for wt HCA II and mutants. Compare the apparent excess specific heat (Cp) vs temperature determined by DSC for (red) Y7I HCA II; (green) Y7F HCA II and (black) wt HCA II.

A B C

Figure 3-5. Crystal structures of the active sites of N62Q and N67Q and Y7F+N67Q HCA II. A) The structure for N67Q HCA II. B) The structure for N62Q HCA II. C) The structure for Y7F+N67Q HCA II. A ball-and-stick diagram of coordinating active site residues as labeled; the zinc ion and the oxygen molecule of waters are shown as gray and red spheres, respectively. The water network of the active-site is labeled W1, W2, etc. Presumed hydrogen bonds are represented as dashed red lines. The conformation of His64 side chain is shown also indicated.

65

CHAPTER 4 PHYSIOLOGIAL ROLE OF HUMAN RED BLOOD CELLS IN THE GENERATION OF

NITRIC OXIDE

The previous two chapters discussed CA on a biophysical level in regard to its

primary physiological reaction of reversible hydration of CO2 to HCO-3 and a proton. A

newly suggested function of CA is a method of NO generation for vasodilation for

signaling in vascular biology [54]. This prompted us to develop a technique with the

membrane inlet mass spectrometer to monitor NO levels in solution. We then applied

this technique to purified deoxygenated hemoglobin (deoxy-Hb(FeII)), and

deoxygenated whole red blood cells.

Although control of basal blood flow in humans is mediated mainly by NO

generated from endothelial nitric oxide synthase (eNOS), considerable research has

focused on other mechanisms that may play a role in vasoregulatory activity, such as

nitrite reductase activity to generate NO. Based on studies showing vasoactivity of

nitrite under hypoxic conditions, increased consumption of nitrite during normoxic

exercise, and positive arterial-to-venous gradients of nitrite, it has been suggested that

the interactions of nitrite with erythrocytes could be a source of vasodilatory NO [55-58].

A prominent and controversial explanation of the vasodilatory effect of nitrite is the

hypothesis that deoxy-Hb(FeII) in erythrocytes produces vasodilation [56, 59, 60]. More

recently the potential nitrite reductase activity of CA was proposed to play a role in the

control of local blood flow that is not dependent on hypoxic or anoxic conditions [54].

Furthermore it has been proposed that the red cell membrane anion exchanger (AE-1)

may help to either mobilize extracellular nitrite for intracellular utilization [61] or facilitate

transmembrane S-nitrosothiol exchange [56], and that aquaporin-1 may act as a gas

channel for NO escape [62]. To understand the role these cells play as a NO source to

66

stimulate vasodilatation the activity of the components needs to be examined

individually.

Generation of NO Catalyzed by CA

CA had only one known physiological function but it has been shown that the

active site can also catalyze the hydrolysis of various ester compounds. Very recently a

report was published introducing for the first time a possible ability of CA to catalyze the

conversion of nitrite into NO. The current proposed mechanism for the generation of

bioavailable NO by carbonic anhydrase does not involve the zinc because its

coordination in the active site does not allow redox activity. CA is proposed to function

as a nitrite anhydrase due to the similar structure and binding to the active site of CA as

bicarbonate. This nitrite anhydrase mechanism is proposed to be equivalent to the

reverse of CO2 hydration described in Chapter 1. The well studied acidic

disproportionation of nitrite involved in the acid-mediated and nitrite-dependent

generation of NO by reactions 1 and 2 below was shown to occur in CA by the Fago

group [54].

- H2O 2NO2

- + 2H+ ⇋ 2HNO2 ⇋ N2O3 (1)

N2O3⇋ NO + NO2 (2)

The generation of protons during the primary mechanism of CO2 hydration may also

increase the acidic disproportionation of nitrite to NO. Interestingly the catalysis by CA

was faster at high pH indicating that CA is not a nitrite reductase [54]. The other

interesting aspect of the Fago study examined the affect of traditional CA inhibitors,

which bind to the zinc (in the same hydrophobic pocket for CO2 binding) on the amount

67

of NO produced. They concluded that the hydration of CO2 generating protons and the

nitrite anhydrase activity may occur simultaneously to produce bioavailable NO [54].

Deoxy-Hemoglobin Mechanism of Nitrite Reductase

There has been great interest in the reactions of nitrite with erythrocytes following

the initial reports of the nitrite reductase activity of deoxy-Hb(FeII) [63]. The reduction of

nitrite by deoxy-Hb(FeII) is a complex, autocatalytic and allosteric reaction that ultimately

results in concentrations of NO-Hb(FeII) and met-Hb(FeIII) that are nearly equal [55, 64].

This is due to the well known one-electron reduction of nitrite by deoxy-Hb(FeII)

resulting in the formation of met-Hb(FeIII). The fate of the resulting NO has been heavily

studied, but it is certain that most of it binds to deoxyHb(FeII).

deoxy-Hb(FeII) + NO2⎯ + H+ NO + met-Hb(FeIII) + OH⎯ (3)

deoxy-Hb(FeII) + NO NO-Hb(FeII) (4)

A significant problem with this scenario is that deoxy-Hb(FeII) avidly binds NO, and this

autocapture is expected to prevent appreciable NO from exiting the erythrocyte. This

effect may be mitigated by generation of an intermediate such as N2O3 which does not

bind tightly to deoxy-Hb(FeII) and could pass across the red cell membrane [60].

Additional data is accumulating that the vasodilatory effect of nitrite is not wholly

related to this nitrite reductase activity in erythrocytes. For example, studies detecting

NO by EPR and chemiluminescence showed that nitrite caused appreciable increases

in NO production in tissues such as liver and heart but only trace amounts in blood [65].

In addition, nitrite reductase activity under anerobic conditions (in vitro) has been

demonstrated for hemoglobin and myoglobin, xanthine oxidase, the bc1 complex of the

mitochondrial electron transport chain, cytochrome P450, and endothelial NO synthase

enzyme [66]. Other studies have suggested a role of S-nitrosothiols as a source of

68

vasodilation, since potent S-nitrosylating agents including N2O3 may be formed from

nitrite [56]. Physiological concentrations of nitrite in humans are in a range up to 1 μM.

The source of nitrite includes foods, the nitrate reductase activity of bacteria in the oral

cavity and GI tract, and eNOS-mediated NO production with subsequent oxidation

which accounts for up to 70% of plasma nitrite [67].

In this report, we examine the reactions of nitrite with CA and other globular

proteins, deoxygenated hemoglobin as well as degassed human erythrocytes using

membrane inlet mass spectrometry to detect the accumulation of NO in the solutions

[68]. In this method an inlet utilizing a silicon rubber membrane is submerged in cell

suspensions and allows NO to pass from extracellular solution into the mass

spectrometer. This provides a direct, continuous, and quantitative determination of nitric

oxide concentrations over long periods without the necessity of purging the suspension

with inert gas. We have not observed accumulation of NO on a physiologically relevant

time scale and conclude that, within the limitations of the mass spectrometric method

and our experimental conditions, erythrocytes do not generate NO within a minute

following the addition of millimolar concentrations of nitrite nor are red cell membrane

anion exchangers and aquaporins critical to the later appearance of extracellular NO.

Methods

Materials

Bovine serum albumin (BSA) and lysozyme were purchased as purified lyophilized

enzymes and resuspended by weight to volume in water to yield 10mg/ml stock

solutions (Sigma-Aldric Saint Louis, MO). HCA II was expressed and purified in the

same manner as the mutant described in Chapter 2. Human blood was freshly collected

and then diluted in isotonic buffer consisting of 50 mM sodium phosphate, 78 mM

69

sodium chloride, 2.7 mM potassium chloride at pH 7.4. Cells were washed by repeated

cycles of centrifugation and dilution in isotonic buffer prior to use. The washed packed

red cells were then diluted into the same isotonic buffer described above at pH 6.7

supplemented with 2 mM of the chelator DTPA (diethylenetriamine-pentaacetic acid

similar to the more common EDTA) within the reaction vessel. The chelator DTPA was

added to prevent metal ion catalyzed nitrite reduction with in the solutions. Degassed

red cells were prepared from these samples by bubbling of helium for 5 minutes prior to

the analysis.

The carbonic anhydrase inhibitors acetazolamide (ACZ) and ethoxyzolamide

(EZA) were obtained from (Sigma-Aldrich, Saint Louis, MO). The compounds 4-

acetamido-4’-isothiocyanstilbene-2,2’-disulfonic acid disodium salt (SITS), and 4,4'-

diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS) were purchased from AnaSpec (San

Jose, CA). Para-chloromercuribenzene sulfonic acid, sodium salt (PCMBS) was

purchased from Toronto Research Chemicals, Inc. The inhibitor 4,4'-dinitrostilbene-2,2'-

disulfonic acid, disodium salt (DNDS) was purchased from Invitrogen Corporation

(Carlsbad, CA).

DIDS, SITS, and DNDS were incubated in red cell suspensions up to 30 minutes

before experiments. Red cells were incubated with PCMBS for 10 minutes prior to

experiments.

Inlet probe and Kinetic Measurements

The membrane inlet consisted of a length of silicon rubber (Silastic) tubing

attached to a piece of glass tubing leading into a dry-ice acetone water trap and then

into an Extrel EXM-200 mass spectrometer, as described elsewhere [68]. This

membrane inlet was immersed in solution contained in a 3 ml glass reaction vessel

70

modified for introduction of samples and inert gas, and sealable by injection septa and

Teflon screw plugs [68]. When immersed in a red cell suspension, this inlet detects free,

unbound, extracellular NO based on the magnitude of the m/z 30 peak (or N2O3 based

on the m/z 76 peak if this species is present). The reaction vessel contained a small

magnetic spinner to continuously mix the reactions. Mass spectra were obtained using

electron impact ionization (70 eV) at an emission current of 1 mA. Source pressures

were approximately 1 x 10-6 torr. The resulting mass scans were well resolved with a

return of ion current (detector response) to the baseline separating each mass unit.

Data collected using the membrane inlet were calibrated by injecting solutions

containing known concentrations of NO into buffered solutions in the reaction vessel

[68]. The sample preparation involved degassing the red cell suspension with helium, as

described above, followed by addition of nitrite through an injection septum. A magnetic

stir bar continuously mixed the suspensions, throughout the experiments.

Results

NO Generation from CA

A few preliminary experiments were done to confirm the observations of Fago by

way of direct measurement of NO generation from CA using the membrane inlet

technique. The addition of nitrite at 8 mM to a solution containing concentrations of HCA

II (1-100 µM) caused a small (2-10 nM) NO accumulation in solution within 10 minutes

(Figure 4-1). The increase in NO generation by CA came to a plateau after an initial

burst of NO generation with the duration dependent on the CA concentration present.

The reactions appeared to plateau well before the majority of the nitrite present (8 mM)

was converted into NO (~10-15 nM). This was compared with the NO accumulation in

solutions containing other globular proteins BSA (~66 kDa) and lysozyme (~14.3 kDa).

71

Similar concentrations of these enzymes created the same level of NO from solutions of

8mM nitrite indicating that this is not an activity specific to CA (Figure 4-2). The addition

of the chelator EDTA to the solution containing 10 µM lysosyme showed that metal ion

contamination could cause the nitrite reduction in these reactions. This is consistent

with the increase in NO generation above the uncatalysed conversion by the addition of

200 µM Fe2+(Figure 4-2).

Additional experiments with HCA II under more physiological conditions (nitrite

concentrations between 1-100 µM) did not show NO generation within the detectable

limit of 1nM. The addition of 40 µM ACZ a classical sulfonamide inhibitor of CA (similar

to those tested by Fago) did not increase the amount of NO generation from the

solutions of CA and nitrite. From these preliminary studies we did not confirm the

presence of nitrite anhydrase activity specific to CA. We propose here a non-specific

reaction of amino acids in globular proteins such as S-nitrosothiol formation that could

be responsible for the amount of NO generated in these experiments.

Deoxy-Hemoglobin Catalyzed Generation of NO

The addition of nitrite at 8 mM to a solution containing 38 µM deoxy-Hb(FeII)

(heme concentration) caused a small lag of about 3 min in which the rate of free,

unbound NO accumulation in solution as detected by the mass spectrometer was small,

followed by a phase of greater rate of accumulation of NO (Figure 4-3). The membrane

inlet method was only detecting free, unbound fraction of the total NO in solution. The

accumulation of NO proceeded to about 14 nM in the experiment using 38 µM deoxy-

Hb(FeII), exceeding the equilibrium level of NO (2 nM) in the absence of deoxy-Hb(FeII).

This level to which [NO] approaches is an exceedingly small fraction (~10−6) of the initial

nitrite added.

72

On addition of nitrite to deoxy-Hb(FeII), both the initial velocity of the appearance

of the NO signal and the plateau level at long times increased with decreasing pH

consistent with a nitrate reductase mechanism. The increase in the initial velocity of NO

formation is consistent with dependence on an ionization with a pKa less than 6.5. This

is possibly the pKa of HNO2 which is near 3.3, although it could represent a pKa on

hemoglobin itself. Increasing initial nitrite concentration increased monotonically the

maximal level of NO at times near 20 min (data not shown). In addition, with increasing

initial nitrite at a constant deoxy Hb(FeII), there was a shortening of the duration of the

initial plateau from over 10 min to less than 2 min at 2 mM to16 mM nitrite respectively

(data not shown).

Human Red Blood Cell Suspensions Generation of NO from Nitrite

We added nitrite at an initial concentration of 2 - 16 mM to a suspension of

degassed, human red cells (0.8% hematocrit) and used the m/z 30 peak to detect

extracellular NO. The appearance of NO was characterized by two phases (Figure 4-

4A). The first phase showed accumulation of extracellular NO forming a low-level

plateau near 1.3 nM, for an initial nitrite concentration of 8.0 mM, with the plateau

lasting over one minute (Figure 4-4B). This initial plateau was slightly less than about 2

nM formed in the uncatalyzed dismutation of an initial concentration of 8.0 mM (Figure

4-4B). The length of the initial plateau decreased with increasing nitrite concentration.

After this initial steady state plateau, there was a second phase in which

extracellular NO concentrations increased and approached a higher plateau of NO

concentration, one that exceeded the concentration of NO in the uncatalyzed reaction.

In this second phase, the rate of increase of NO and level of the apparent second

plateau increased with increasing concentrations of nitrite (Figure 4-4A). The data

73

shown in Figure 4-4 are for human red cells that were washed three times in isotonic

buffer; however, the results were identical when an equivalent amount of blood was

used without prior washing of the red cells. These data can be compared with the

accumulation of NO in solutions of hemoglobin (Figure 4-3) [69].

When the pH of the external solution was decreased, we observed a decrease in

the length of the first plateau accompanied by an increase in the rate of appearance of

extracellular NO and apparent plateau level for the second phase (Figure 4-5). A

qualitatively similar effect was also observed in solutions of deoxyhemoglobin [69] and

is related to the concentration of HNO2, the conjugate acid of nitrite with a pKa of 3.3. As

with solutions of deoxyhemoglobin [69], the logarithm of the rates of NO accumulation in

the second phase was a linear function of pH (R2 = 0.97) with rates increasing as pH

decreases.

We extended these studies to suspensions of larger hematocrit using additions of

8 mM nitrite. Increasing the amount of degassed erythrocytes increased the length of

the initial plateau, and in the approach to the second plateau decreased the rate of

extracellular NO accumulation and the level of the second plateau itself (Figure 4-6).

We did not observe in these experiments an increase in the peak at m/z 76 that would

correspond to the accumulation of N2O3. The generation of NO in conditions closer to

physiological was then examined. We used human, degassed erythrocyte suspensions

at 50% hematocrit in isotonic buffer at pH 7.4 and 37 °C. The suspension was then

injected with an initial 1.0 mM nitrite concentration and monitored for NO generation

extracellularly. These conditions did not generate any detectable NO for as long as 30

minutes following the addition of the nitrite.

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Additional experiments were performed at varying levels of oxygenation under the

conditions of Figure 4-4. Mixing oxygenated and degassed packed red cells in different

ratios was used to produce different levels of oxygenation. Completely oxygenated red

cell suspensions showed no accumulation of NO upon addition of nitrite. Intermediate

levels of oxygenation showed the two-stage accumulation of NO as in Figure 4-4 with

NO concentrations which were intermediate between those observed for completely

deoxygenated and oxygenated red cells.

Effect of EAZ on NO Accumulation

We tested a classical inhibitor of CA for the effect on the accumulation of

extracellular NO after introduction of nitrite to degassed whole red cell suspenctions.

Concentrations of EZA from 1 μM to 1mM had no effect on the accumulation of NO in

extracellular fluid, shown for three concentrations of EZA in Figure 4-7. EZA, which

readily crosses the membrane, blocks all CA activity in these experiments [70]. In

control experiments, EZA in the concentration ranges used here caused no change in

the rate of accumulation of NO in solutions containing deoxyhemoglobin but no

erythrocytes. These results were similar to those performed with purified CA and ACZ

(which is slowly membrane permeable) indicating that the inhibition of CA does not

increase the amount of NO generation.

Effect of DIDS on NO Accumulation

We tested several inhibitors of the band 3 anion exchanger (AE-1) for their effect

on the accumulation of extracellular NO after introduction of nitrite. Concentrations of

DIDS from 0.1 μM to 1.0 mM had no effect on the accumulation of NO in extracellular

fluid, shown for four concentrations of DIDS in Figure 4-8. An apparent IC50 of 0.1 μM

DIDS was reported for the inhibition of the transport of sulfate by the anion exchangers

75

in human red cells [70]. Results identical to Figure 4-8 were obtained when experiments

were performed in the dark to prevent light induced isomerization of DIDS [71]. In

additional experiments, we used solutions of DIDS containing potassium bicarbonate

(0.1 mM) or DMSO (5% in final suspensions) to prevent the formation of polymeric

DIDS [15]; results were identical to Figure 4-5.

Experiments under the same conditions using the anion exchange inhibitors SITS

and DNDS also showed no effect in the accumulation of NO (data not shown). SITS

was examined in the concentration range from 10 μM to 100 μM. An IC 50 of 50 μM for

SITS is reported for the inhibition of the transport of sulfate by the anion exchangers in

human red cells [72]. DNDS was used at concentrations above and below its IC50 of 2

uM for inhibition of sulfate transport through the anion exchange protein of human red

cells [70]. In control experiments, DIDS, SITS, DNDS in the concentration ranges used

here caused no change in the rate of accumulation of NO in solutions containing

deoxyhemoglobin but no erythrocytes.

Effect of PCMBS on NO Accumulation

We added to our red cell suspensions PCMBS which is an inhibitor with complex

effects on red cells including inhibition of the aquaporin-1 channel but also inhibition of

anion exchange proteins to some extent [73]. The concentrations of PCMBS used in our

studies are in the range of the inhibition of aquaporin demonstrated by the reduction of

water diffusion by half at concentrations of 2mM in adult human erythrocytes [74]. The

reported IC 50 of 0.5 mM for PCMBS is reported for the inhibition of CO2 transport

through the aquaporin 1 in human red cells [75] . A range of PCMBS from 25 μM to 2

mM was used in our studies; this range included both inhibition of the aquaporin

channel as well as inhibition of the band 3 anion exchanger (IC50 near 2 mM) [73, 75].

76

The presence of PCMBS showed no significant change in the level of NO for the

initial plateau (up to 3 minutes) after addition of nitrite (Figure 4-9). For the second

phase, when 25 μM or 50 μM PCMBS was incubated with the red cells, there was an

increase in plateau level (Figure 4-9). At the higher PCMBS concentration of 1 mM and

2 mM the second plateau appeared lower than in the absence of PCMBS, which was

observed in three repetitions of the experiment. PCMBS did not affect the rate of

accumulation of NO in solutions containing deoxyhemoglobin but no erythrocytes at

concentrations below 50 μM.

Discussion

In measuring the accumulation of NO in red cell suspensions by membrane inlet

mass spectrometry, the inlet itself is immersed in the suspension. Hence, the free,

unbound NO in the extracellular fluid is measured without purging the suspension with

inert gas, giving a direct and real time measure of NO concentration in the extracellular

solution. We have observed a biphasic increase in NO measured in this manner after

adding millimolar levels of nitrite to human, degassed erythrocyte suspensions (Figure

4-4A,B). The uncatalyzed dismutation of nitrite observed by this method was reported

earlier [68] and is shown in Figure 4-4B in comparison with data obtained using

erythrocyte suspensions, and in Figure 4-3 in solutions containing hemoglobin. The

topic of the uncatalyzed dismutation of nitrite is covered in a classic review from the

1950’s [76] which has been followed by numerous reports of the rate constants for this

complex process [77, 78].

Accumulation of Extracellular NO in Red Cell Suspensions

The very low level of NO in the initial 1 - 2 minutes after addition of nitrite to

degassed erythrocyte suspensions is consistent with the tight binding of NO to

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deoxyHb(FeII) in the nitrite reductase scheme (eqs. (1) and (2)) in the interior of red

blood cells. Even at the very large concentration of initial nitrite at 2 mM and 4 mM, the

levels of NO detected in the extracellular fluid do not exceed 1 nM (Figure 4-4B) for at

least five minutes, below the level of NO consistent with vasodilation (1 to 10 nM).

Figure 4-4B shows that for the addition of 8 mM nitrite the concentrations of

extracellular NO were somewhat lower than the control without red cells, consistent with

the suggestion that NO generated is taken up by the red cells. This is particularly

evident at the larger hematocrits shown in Figure 4-6 in which the concentration of NO

outside of the cells decreases significantly when there are more erythrocytes present.

Hypoxia during ischemia would be conducive to additional generation of NO by the red

cells as the decrease in pH results in quantitative increases of NO generation in the red

cell suspensions (Figure 4-5). The reduction in pH increases HNO2 concentrations and

this may be the predominant species entering the red cell to be reduced to NO.

Nitrite concentrations used at 2 to 16 mM differ greatly from physiological

concentrations of nitrite, which are near 1 μM; however, the much larger concentrations

of nitrite needed to observe NO in red cell suspensions forms part of our conclusion.

Addition of 1.0 mM nitrite under physiological conditions did not show any accumulation

of NO that we could measure. For the membrane inlet that we have used the half-time

is near 5 seconds for response to step increases in concentration of NO. We would not

be able to observe a small, rapid pulse of NO increase occurring within seconds after

the addition of nitrite.

In these red cell suspension, the low-level plateau of the first phase (up to 2-3

minutes) represents a complex steady state in extracellular NO concentration involving

78

several processes including the uncatalyzed dismutation of nitrite, the flux of nitrite and

HNO2 into the cells where it reacts with hemoglobin according to eqs. (1) and (2), and

the diffusion of NO across the cell membrane. There are a number of other

accompanying processes such as nitrosylation reactions, the possible role of other

species of nitrogen oxides, other reactions of nitrite and NO in the cell, and the action of

metHb(FeIII) reductase.

The second phase is an increase in the rate of extracellular NO accumulation that

follows the initial plateau and occurs many minutes after addition of nitrite to degassed

erythrocyte suspensions (Figure 4-4A,B). The cause of this second phase of increase

and apparent second plateau is unclear, but we believe it is related to the slow off rate

of NO from the nitroso-hemoglobin complex NOHb(FeII) generating free NO which then

exits the cell since nearly all deoxyHb(FeII) is bound by NO [69]. This dissociation also

generates deoxyhemoglobin that is again available for the reactions of Eqs. (1) and (2).

The resulting progress curve for the appearance of NO in extracellular solution after

addition of nitrite (Figure 4-4A) is very similar to that obtained by this method under

similar conditions for solutions containing deoxyhemoglobin (Figure 4-3) [69]. Both

experiments, using degassed erythrocyte suspensions or solutions of deoxyHb(FeII),

showed an initial phase of low levels of NO and a second phase of more rapid NO

accumulation approaching a plateau. Moreover the time scales and magnitudes of NO

observed are similar for the red cell and solution phase studies with purified hemoglobin

(Figures 4-3,4-4). These data show upon introduction of large concentrations of nitrite to

degassed suspensions of red cells, the levels of extracellular NO observed almost

entirely reflect the reactions of nitrite with deoxyhemoglobin in the cells.

79

Inhibition of Band 3 Anion Exchanger of Red Cells

The exchange protein AE-1 is a member of a family of anion exchangers that is

located in the plasma membrane of erythrocytes as well as other tissues and is involved

in the transport of CO2 and bicarbonate in respiration [79, 80]. Studies with intact red

cells and ghosts show nitrite uptake blocked by DIDS, an inhibitor of anion exchange

proteins including AE-1 [81]. However, other studies show no effect of DIDS on nitrite

induced formation of met-Hb(FeIII) in erythrocytes [82, 83]. There will be some diffusion

of HNO2 (pKa 3.3) across the membrane, and studies indicate some uptake of nitrite by

the sodium-dependent phosphate transporter [83].

We observed no significant change caused by the inhibitors of anion exchange

DIDS, SITS, or DNDS in the rate of extracellular NO accumulation in this work (shown

for DIDS in Figure 4-8). The disulfonic stilbene compounds including DIDS, SITS, and

DNDS are a class of the most potent and commonly used inhibitors of the exchange

protein AE-1. These inhibitors in the concentration range of their respective IC50 values

did not affect NO accumulation under conditions of Figure 4-4.

These results show that anion flux using the exchange protein AE-1 is not a

predominant factor in the accumulation of extracellular NO caused by addition of large

quantities of extracellular nitrite. Straightforward computational simulations

(www.polymath-software.com) of these nitrite reactions with red cells suggest that the

flux of HNO2 into the cells under the conditions of Figure 4-9 may play an important role

under our conditions, so that inhibition of the band 3 anion exchanger does not make a

significant contribution to the NO levels we observed.

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Inhibition of Aquaporin-1 Channel of Red Cells

Being a small uncharged molecule, NO is expected to pass readily across

membranes [84], and there is evidence that some passes through the aquaporin

channel [62]. Evidence that a large amount of CO2 fluxes out of red cells through the

aquaporin channel suggests that it may be a conduit for NO and other dissolved gases

to enter or exit the red cell, although the significance of this is uncertain [75].

PCMBS had no effect on the levels of extracellular NO in the initial plateau after

addition of nitrite (Figure 4-9). In addition, PCMBS at the concentrations of Figure 4-9

had no effect on the accumulation of NO in solutions of deoxyhemoglobin (data not

shown). These data show that the aquaporin channel does not play a predominant role

in the complex steady-state processes that are involved in the initial response of red

cells to large concentrations of nitrite. Rather, the steady state appears dominated by

the flux of NO through other pathways and of HNO2 followed by the autocapture of NO

in the red cells. The complex behavior of PCMBS toward the second plateau in Figure

4-9 is unexplained at present. We can conclude however that flux through the aquaporin

channel is not a major factor for accumulation of extracellular NO in our experiments.

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Figure 4-1. NO accumulation from nitrite at various concentrations of HCA II. The generation of NO from addition of 8mM sodium nitrite to solutions of (green) 1.0 μM HCA II; (red) 10 μM HCA II; (blue) 100 μM HCA II; and (black) control containing no enzyme. Solutions contained 50 mM phosphate and 2 mM DTPA (diethylenetriaminepentaacetic acid) at pH 6.6 and 25 °C.

82

Figure 4-2. NO accumulation from various globular proteins and metal ion

contamination. The generation of NO from addition of 8mM sodium nitrite to solutions of (green) 1.0 μM BSA; (red) 10 μM BSA; (purple) 10 μM losozyme; (blue) 200 μM Fe2+; and (black) control containing no enzyme. Arrow indications the addition of EDTA to a final concentration of 20 mM after 10 minutes to the lysozyme solution. Solutions contained 50 mM phosphate and 2 mM DTPA (diethylenetriaminepentaacetic acid) at pH 6.6 and 25 °C.

83

Figure 4-3. The time course of NO accumulation from various forms of hemoglobin. The time course of dissolved NO concentrations (obtained from m/z 30) upon addition of nitrite to solutions containing (blue) 38 μM deoxy-Hb(FeII); or (red) 38 μM oxy-Hb(FeII); or (black) no hemoglobin. (These are heme concentrations.) At time zero, NaNO2 was added to attain a concentration of 8 mM. Solutions also contained 50 mM phosphate buffer at pH 6.8, 110 mM NaCl, 2 mM EDTA at 23 °C. These data from ref [69].

84

A B Figure 4-4. Extracellular NO accumulation obtained from a suspension of degassed

human red cells. A) Time course of extracellular NO accumulation obtained from m/z 30 detected by membrane inlet mass spectrometry upon addition of sodium nitrite to a suspension degassed human red cells at 0.8% hematocrit. At time zero, NaNO2 was added at the concentrations identified on the plot. Solutions also contained 50 mM sodium phosphate, 78 mM sodium chloride, 2.7 mM potassium chloride, and 2 mM DTPA at pH 6.7 and 25 °C. B) The same series of experiments showing an expansion of the early times. The dotted line represents the NO generated by addition of NaNO2 at a concentration of 8.0 mM to the identical solution but containing no red cells.

85

Figure 4-5. The pH dependence of extracellular NO accumulation. The dependence of

the accumulation of extracellular NO upon addition of sodium nitrite to a suspension of degassed human red cells at 0.8% hematocrit from pH 6.4 to 7.4. At time zero, NaNO2 was added at a concentration of 8.0 mM at the pH identified on the plot. Other conditions were as described in the legend to Figure 4-4.

86

Figure 4-6. The dependence on hematocrit of extracellular NO accumulation. The dependence of the accumulation of extracellular NO on the amount of human, degassed erythrocytes in suspensions. At time zero, NaNO2 was added at an initial concentration of 8.0 mM to the suspension containing 25 μL, 250 μL, 625 μL, or 1250 μL packed red cell volumes to the final hematocrits identified on the plot. Other conditions were as described in the legend to Figure 4-4.

87

Figure 4-7. The effect of EZA on extracellular NO by degassed human erythrocytes. Effect of EZA, an inhibitor of CA, on accumulation of extracellular NO in suspensions of degassed human erythrocytes at 1.6% hematocrit. In each experiment 8.0 mM NaNO2 was added at time zero to a suspension containing the designated concentrations of EZA: (green) 10 μM; (red) 100 μM; (yellow) 1000 μM; and (blue) no EZA. In each case, EZA was incubated with erythrocytes 5 minutes prior to addition of nitrite. The other conditions were as described in the legend to Figure 4-4.

88

Figure 4-8. The effect of DIDS on extracellular NO by degassed human erythrocytes.

The effect of DIDS, an inhibitor of anion exchange, on accumulation of extracellular NO in suspensions of degassed human erythrocytes at 1.6% hematocrit. In each experiment 8.0 mM NaNO2 was added at time zero to a suspension containing the designated concentrations of DIDS: (black) 0.1 μM; (green) 1 μM; (purple) 10 μM; (red) 100 μM; and (blue) no DIDS. In each case, DIDS was incubated with erythrocytes 10 minutes prior to addition of nitrite. The other conditions were as described in the legend to Figure 4-4.

89

90

Figure 4-9. The effect of PCMBS on extracellular NO by degassed human erythrocytes.

Effect of PCMBS, an inhibitor of the aquaporin channel, on the accumulation of extracellular NO concentration (determined from m/z 30) in suspensions of human erythrocytes at 0.8% hematocrit following the addition of an initial concentration of 8.0 mM nitrite. Suspensions contained the following concentrations of PCMBS: (red) 25 μM; (green) 50 μM; (black) 100 μM; (blue) no PCMBS: (brown) 1 mM; and (purple) 2 mM labeled A-F respectively on the plot. The data in grey labeled G show the accumulation of NO after addition of 8.0 mM nitrite in solutions containing no erythrocytes. Other conditions were as described in the legend to Figure 4-4.

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

The aim of the dissertation was to gain insight into the functions of CA by studying

both the kinetics and the structural features that promote these functions. Specifically

we examined the rates of proton transfer in HCA II through a series of mutations in key

hydrophilic residues in the extended active site (Chapter 2) and their resulting structural

changes (Chapter 3). Additionally the possible new CA function (and other proteins

highly abundant in erythrocytes) to generate NO that could signal increased

vasodilatation in the vasculature was examined (Chapter 4).

The mutational analysis was designed to manipulate the water network and the

conformation and flexibility of the intramolecular proton shuttle residue His64. The

residues 7,62, and 67 were chosen for being in the hydrophilic region of the active site

within hydrogen boding distance to the branching waters (W3a and W3b) and near

His64. The attempt was to compile these mutational analyses with the wide body of

evidence on this mutant already established to separate the influences of the three

factors that appear to control the rate of proton transfer: the orientation of His64, the

water network connecting the proton donor and acceptor, and the pKa values of the

proton donor and acceptor. The relationship between structure and function could then

be applied to understanding proton transfer in more complex biological systems.

The compilation of the kinetic and structural changes in these mutants of HCA II

provides insights about those three factors. Mutations in this hydrophilic region

increased the proton transfer rates over those observed in wild type up to 15 fold. A

range of increases were observed which indicated the hydrophilic region is essential to

tune the pKa of His64. Increases in kB correlate with decreased pKa of His64 providing

91

more evidence that the acidic nature of the imidazolium ion transfers protons through a

network in catalysis. The inward orientation of His64 was associated with a decrease in

the distance a proton must travel to reach the zinc-bound hydroxide. The inward

conformation of His64 makes a stronger hydrogen bond to W2 in Y7F and more so in

the Y7F+N67Q mutant. While both N62Q and N67Q had a majority of His64 being in the

outward conformation; the active site preformed proton transfer at rates near wild type.

Kinetically the changes in kB followed Marcus theory to a large extent for most of the Y7

variants indicating changes in the ΔpKa values can predict 2-4 fold changes in proton

transfer rates in these cases. Changes greater than 5 fold for Y7F and the double

mutant could be caused by a synergism that created a short, single stranded water

network between the proton donor and acceptor.

The ability to observe ordered water in the active-site cavity through crystal

structure has provided significant insight to the role of rapid proton transfer despite the

picosecond lifetime of these networks in solution. Computations show a more rapid

proton transfer through a single, non-branched chain of water molecules compared with

branched chains [30, 52]. The Y7F and Y7F+N67Q HCA II variants were consistent with

the suggestion that efficient proton transfer is enhanced more by a single water strand

than it is by changes in ΔpKa. The N62Q mutant contained an additional water molecule

which further branches the water network and falls below the value predicted by

changes only in ΔpKa. The current concept of proton transfer energetics is drawn from

primarily from experimental evidence certainly that emphasize the formation of the

water chain connecting donor and acceptor as a precondition for proton transfer. This is

further supported by the stronger hydrogen bonds between zinc bound solvent, W1,

92

W2, and His64 producing her fastest rate of proton transfer yet observe 12.3 μs-1 in

Y7F+N67Q HCA II. However, the relevance of the ordered water structure observed in

the crystal structures of the enzymes compared to solution is still uncertain.

Yet another consideration is the energy barrier to proton transfer between the zinc-

bound solvent molecule and His64 that is entirely inward or entirely outward. Multistate

empirical valence bond computations show the energy barrier for proton transfer

between outward His64 and inward is only about 6 kcal/mol [45]. This is less than

approximately 8-10 kcal/mol for the proton transfer. Here we observed a mixed

population of completely inward and outward occurring His64, yet all the mutants have

rates of proton transfer faster than wild type. This raises the point that in solution the low

barrier between in and out conformation of His64 may not change the rate of proton

transfer. However, the data on the very fast Y7F+N67Q HCA II show the shortest

overall distance of 6.7 Å is associated with faster proton transport. This result is in

agreement with a similar study that replaced His64 with an alanine and then added a

histidine residue in different locations of the active site which showed the distance

between 7.5 and 6.6 Å to allow proton transfer [85]. Similarly the relevance of the

orientation of His64 observed in the crystal structures may not truly reflect the

population of inward and outward conformation in solution.

A particularly interesting feature of the structural examination of Y7I HCA II was

the significant change in the N-terminal residues which has never been observed in

HCA II structures before. This indicated that Tyr7 may help the other aromatic residues

of the N-terminus to fold into the compact protein. The decrease in thermal stability

observed in the Tyr7 mutants indicated, despite their increase in proton transfer that

93

these proteins would probably not be physiologically selected. The Y7I mutant may also

be helpful in the studies to determine the conformation of the N-terminus in binding to

the AE-1. The possibility of a metabolon within human red cells between the

bicarbonate transporting protein AE-1and CA generating bicarbonate is still being

examined.

There are still many unanswered questions about the proton transfer pathway in

HCA II. Despite recent high resolution x-ray structures and neutron structure of HCA II

there are still questions about which active site residues directly participate in hydrogen

bond interactions with water molecules and which are hydrogen bond donors and

acceptors involved in proton transfer. The roles of Thr199 and Thr200 hydrogen

bonding to zinc-bound solvent, W1 and the importance of a deep water molecule have

been clearer from these studies in the conversion between CO2 and bicarbonate. On

the other hand, the conformation of His64 and the role of Tyr7 in orienting the water

network are still unclear. There does not appear to be a simple method to examine the

factors which influence proton transfer individually within a series of active site

mutations. In many cases, a combination of several factors together regulate the rate of

proton transfer, as in the synergism observed between the Y7F and N67Q mutants

acting together in a non-additive manner to increase the rate constant for proton

transfer.

These questions can be re-examined in the light of the kinetics and structures

determined here in by molecular dynamics groups. These data provided information on

the contribution of the proton shuttle His64 as well the water networks in the rate of

proton transfer. These studies contribute to our current understanding of the CA active

94

95

site and catalysis, and what contributes to facilitate proton transfer in protein

environments. Advancing the theoretical understanding of proton transfer is important

for understanding the proton transfer processes of many other proteins including ATP

synthase, rhodopsin, and the photosynthetic reaction center. The conservative

substitutions (such as N62Q and N67Q) can also be used to understand differences

between human isoforms which may be useful for isoform specific drug design.

The examination of potential NO generating reactions by both carbonic anhydrase

and hemoglobin individually and in the context of whole red blood cell suspensions was

reported in Chapter 4. The application of membrane inlet mass spectrometry showed an

initial burst of NO by addition of millimolar levels of nitrite to CA solutions but did not

appear to generate NO in a specific manner. The preliminary studies on CA suggest a

more careful examination of physiological conditions to determine if the NO generated

was catalyzed by CA or a non-specific acidic-base mechanism or from contaminating

metals. The application of membrane inlet mass spectrometry showed no initial burst of

extracellular NO or N2O3 caused by addition of millimolar levels of nitrite to suspensions

of degassed red cells at pH 6.7. Erythrocytes do not generate NO above the

uncatalyzed level within a minute following the addition of millimolar concentrations of

nitrite nor are CA, red cell membrane AE-1, or aquaporins critical to the later

appearance of extracellular NO. These data support the hypothesis that, although the

nitrite reductase reactions of deoxyHb(FeII) apply, the autocapture of NO by

deoxyHb(FeII) precludes efflux of NO. In the controversy over nitrite reductase activity,

these conclusions are consistent with no role of red cells in the mechanisms of NO

generation from nitrite to stimulate vasodilatation.

LIST OF REFERENCES

[1] Silverman, D. N.; Lindskog, S. The catalytic mechanism of carbonic anhydrase: implications of a rate-limiting protolysis of water. Accounts of Chemical Research 21:30-36; 1988.

[2] Hewett-Emmett, D.; Tashian, R. E. Functional Diversity, Conservation, and Convergence in the Evolution of the [alpha]-, [beta]-, and [gamma]-Carbonic Anhydrase Gene Families. Molecular Phylogenetics and Evolution 5:50-77; 1996.

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BIOGRAPHICAL SKETCH

Rose Lynn Mikulski was born in Sunrise, Fl in 1983. She attended Ely High

School, graduating in 2002. She immediately entered the undergraduate program at

University of Florida and graduated in 2006 with a B.S. in Microbiology, a B.A. in

political science and a minor in chemistry. She spent her senior year studying the

interface of MnSOD proteins from E.coli and Humans under the guidance of Dr. Robert

McKenna. In the fall of 2006, Rose began her graduate school training in the University

of Florida’s Interdisciplinary Program in biomedical sciences. She joined the lab of Dr.

David Silverman in the spring of 2006 to research the catalysis of human carbonic

anhydrase II and a possible physiological mechanism for nitric oxide generation in

human red blood cells. She was awarded a grant in 2008 by the University of Florida’s

Medical Guild to measure directly the NO generation from interactions of nitrite with

human red cells. She was later awarded an American Heart Association predoctoral

fellowship in 2009 to continue examining the mass spectrometric measurement of nitric

oxide.

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