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Kinetics and inhibition of carbonic anhydrase III, comparison with carbonic anhydrase II

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Title:
Kinetics and inhibition of carbonic anhydrase III, comparison with carbonic anhydrase II
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Kararli, Tugrul T., 1956-
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English
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viii, 106 leaves : ill. ; 29 cm.

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Active sites ( jstor )
Anions ( jstor )
Catalysis ( jstor )
Enzymes ( jstor )
Fluorescence ( jstor )
Imidazoles ( jstor )
Kinetics ( jstor )
pH ( jstor )
Protons ( jstor )
Sulfonamides ( jstor )
Carbonic Anhydrases ( mesh )
Carbonic anhydrase inhibitors ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF ( mesh )
Pharmacology and Therapeutics thesis Ph.D ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 100-105.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Tugrul T. Kararli.

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KINETICS AND INHIBITION OF CARBONIC ANHYDRASE III:
COMPARISON WITH CARBONIC ANHYDRASE II





By



TUGRUL T. KARARLI


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

UNIVERSITY OF FLORIDA


1984




KINETICS AND INHIBITION OF CARBONIC ANHYDRASE III
COMPARISON WITH CARBONIC ANHYDRASE II
By
TUGRUL T. KARARLI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984


This dissertation is dedicated to my parents, Fatma and Mehmet,
whose love, support and sacrifices have brought me to where I am.


ACKNOWLEDGMENTS
I am grateful to Dr. David N. Silverman for his excellent guidance
throughout my graduate education to become an independent scientist.
I would like to thank Dr. C. K. Tu for helping me with his valuable
technical expertise and thoughtful advice in my research problems. I
acknowledge Dr. R. Rowlett with whom I have had valuable discussions.
I would like to extend my gratitude to Mr. Dave Godman for his very
valuable help with the perfusion techniques. I would like to thank
Dr. W. Kern, Dr. Gautam Sanya!, Mr. George C. Wynns and Mr. Ness Pessah
for their contributions in protein purification. I especially
acknowledge Mrs. Judy Adams for her excellent job of typing this
dissertation. Finally, I would like to acknowledge the Scientific and
Technical Research Council of Turkey for their financial support
during my doctoral education.


PREFACE
This dissertation is composed of an introductory chapter, four
chapters written in standard manuscript style, a conclusions chapter
and an appendix section. Due to this format, some material may appear
redundant. It is hoped that the reader will accept the repeated
material as being important in the scope of this dissertation.
IV


TABLE OF CONTENTS
PAGE
ACKNOWLEDGMENTS iii
PREFACE iv
ABSTRACT vii
CHAPTER ONE INTRODUCTION 1
CHAPTER TWO INHIBITION BY CUPRIC IONS OF HYDRATION OF CO2
AND DEHYDRATION OF HCO3 CATALYZED BY HUMAN
CARBONIC ANHYDRASE II 8
Introduction 8
Experimental Procedure 9
Results 13
Discussion 23
CHAPTER THREE HYDRATION, DEHYDRATION AND ESTERASE ACTIVITY
OF CAT CARBONIC ANHYDRASE III 29
Introduction 29
Experimental Procedure 30
Results 33
Discussion 40
CHAPTER FOUR RATE-CONTRIBUTING EVENTS IN THE HYDRATION
OF CO2 CATALYZED BY CARBONIC ANHYDRASE III
FROM CAT SKELETAL MUSCLE 45
Introduction 45
Experimental Procedure 46
Results 46
Discussion 53
CHAPTER FIVE INTERACTION OF CAT CARBONIC ANHYDRASE III
WITH ANIONS AND SULFONAMIDES 59
Introduction 59
Experimental Procedure 60
Results 62
Discussion 75
CHAPTER SIX CONCLUSIONS 81
v


Table of Contents--Continued
APPENDIX A 83
APPENDIX B 90
REFERENCES 100
BIOGRAPHICAL SKETCH 106
vi


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
KINETICS AND INHIBITION OF CARBONIC ANHYDRASE III:
COMPARISON WITH CARBONIC ANHYDRASE II
By
Tugrul T. Kararli
December, 1984
Chairman: David N. Silverman
Major Department: Pharmacology and Therapeutics
In order to get more information about the rate-contributing
intramolecular proton transfer step in the catalytic mechanism of
carbonic anhydrase II, the inhibition by Cu^+ of the hydration of CO2
and dehydration of HCO3 catalyzed by human isozyme II was measured over
the pH range 6.0 to 7.6 by using a stopped-flow technique. Also, the
interaction of Cu~+ with the active site sphere of human carbonic
anhydrase II was evaluated by a fluorescence method which utilizes
fluorescent dansylsulfonamide. The inhibition of both hydration and
dehydration reaction by Cu were mixed/noncompetitive at a fixed high
buffer concentration. The corresponding pattern was uncompetitive v/hen
concentration of CO2 was fixed and buffer varied. The inhibition
constant for Cu 'was affected by a group with a p! These results, which were also verified by simulation of the catalysis
pi
with computer methods, suggest that Cu binds to His 64 of carbonic
anhydrase II and inhibits an intramolecular proton transfer.
VI 1


To determine the kinetic mechanism of isozyme III, several kinetic
parameters of this isozyme from cat were measured by using stopped-
flow, fluorescence, 0 exchange and spectrophotometric techniques. The
CO?
pH profiles of kcat and kenz were consistent with the isozyme having a
zinc-bound H2O with a pKa < 6 and a basic residue with a pKa^9.
HCO3 HCO3
Increasing values of kcat /Km as pH was decreased, also supported such
C02
a low pKa for zinc-bound H2O. The isotope effects of magnitude 2.6 in kcat
C02 C02
and 1.3 in kcat/Km were indicative of a rate-limiting proton transfer
transfer located outside the interconversion of C02 and HCO3. Buffers
did not have any affect in the catalysis of C02 by isozyme III. The uncom
petitive/mixed patterns of inhibition of the hydration reaction at low as
well as at high pH by N3 and I- further supported a pKa < 6 for zinc-
bound H20 and a rate-limiting event outside the interconversion of C02
and HCO3. Inhibition constants of N3 and binding constant of
chlorzolamide were dependent on the ionization of a group with a pka > 8.
Anions and sulfonamides were shown to compete for the same or an overlapping
binding site on isozyme III.
vi 11


CHAPTER ONE
INTRODUCTION
Carbonic anhydrase is a zinc-containing metalloenzyme which
catalyzes the reversible hydration of C02 as given in equation 1.
C02 + H20-t~^ HCO3 + H+ (1)
In this process, there is a net release of one proton and the HCO3
ion. The reaction 1 is catalyzed by all three mammalian carbonic
anhydrase isozymes and follows the Michaelis-Menten kinetics. The
turnover number of this reaction for each isozyme is given in Table 1.
The understanding of the catalytic groups and rate-limiting events in
the kinetic mechanism of carbonic anhydrase II has been a main focus of
intense research in the past. (See Appendix A for history, distribution
and general properties of isozymes I and II.)
Steiner eit al_. (1) for the human and Pocker and Bjorkquist (2) for
the bovine isozyme II found that while the Michaelis-Menten constants
C02 HC05
Km and Km for the forward and reverse reactions are independent of
C02 HCO3
pH, the turnover numbers kcai- and kcat are correspondingly dependent
on the basic and acidic form of an activity controlling group titrating
with a pKa close to 7. Experimental evidence suggests that this
group is the water molecule on the zinc (3). It was shown by Jonnson
et aj_. (5) at steady state and by Silverman and Tu (6) at chemical
equilibrium that reaction 1 catalyzed by isozyme II is limited by the
release of the product proton to the solution in the absence of or at
low concentration of buffers. Addition of buffers up to 5-10 mM
1


2
TABLE 1
MICHAELIS-MENTEN PARAMETERS FOR THE ISOZYMES OF CARBONIC ANHYDRASE
co2
kcat (sec-1)
C02
Km (mM)
C02 C02
^cat/^m
(Ml secl)
Isozyme Ia
1.2 x 105
4.2
2.9 x 107
Isozyme 11^
1.0 x 106
8.3
1.2 x 108
Isozyme 11Ic
1.0 x 104
30.0
3.3 x 105
a,^Data from the high pH (pH > 8) of Lindskog et al_. (7) (25C).
c0ata from this study at pH 9.5 and 25C.


3
concentration enhances the rates of reaction 1 by increasing the rate
of this proton release (6). Understanding of the rate-determining
events in the presence of high buffer concentration came from the
measurement of the rates of reaction 1 catalyzed by carbonic anhydrase
II in H2O and D2O. Again, Steiner et al_. (1) for the human and Pocker
and Bjorkquist (2) for the bovine carbonic anhydrase II found that
C02 C02
while kcgt and Km have isotope effects of magnitude as big as 3 to 4,
COo C02
kcat'Km has an isotope effect of unity. A similar pattern was also
observed for the reverse reaction in these studies. All the reaction
- CO2 CO2
steps occurring before the departure of HCO3 are included in ^cat^rn *
C02 C02
This isotope effect of unity on kca|/Km lead Steiner et jiK (1) to
propose the enzymic mechanism given in Scheme I, with its
rate-contributing intramolecular proton transfer located outside the
CO2-HCO3 interconversion. There is some experimental evidence to
o
suggest that histidine 64 residue of isozyme II which is located 6 A
away from the zinc (8) shuttles protons between the catalytic group and
solvent in this rate-contributing event.
Later, 180 exchange experiments by Sil verman et jil_. (9) and Tu and
Silverman (10) at chemical equilibrium have been very useful in further
1 p
understanding of this rate-contributing proton transfer. In the 0
exchange technique, one can measure the rate of the CO2-HCO3
exch COp
interconversion, kcat /Keff, separate from rate of the release of
^-labelled ligand water from the active site, k^o* Measurement of
exch C02
these rates in I^O and D2O showed that while kcat /Keff does not
have any isotope effect, k^o *ias an isotope effect of magnitude as big
as 8.0 at pH 6.6 (9,10). Tu et al. (11) further showed that Cu^+ is a


Scheme 1. Proposed mechanism of human carbonic anhdyrase II
catalysis. EX refers to the transitory complexes ES and
EP, and HEX refers to HES and HEP, respectively. S and P
stand for CO2 and HCO3. Numerals indicate the steps to
which rate constants with the corresponding subscripts
refer. H following E (as in EH) denotes the enzyme with a
protonated activity-controlling group, probably a
zinc-bound water molecule. H preceding E (as in HEX)
denotes the enzyme with a protonated group involved in the
shuttling of protons between the activity-controlling
group and the external medium. Histidine-63 is a likely
proton shuttle group. (With permission from Dr. Roger
Rowlett.)


LO


fa
specific inhibitor of this intramolecular proton transfer with an IC^q
value of 1 x 10^ M at pH 7.3. All these results are certainly in
favor of the catalytic mechanism of isozyme II given in Scheme I.
After 50 years since its discovery, there are still some unanswered
questions about the kinetic mechanism of carbonic anhydrase II. One
is the mechanism of the intramolecular proton transfer, especially the
group responsible for it. For this purpose, I have chosen Cu^+ as a
tool and measured its inhibition of the hydration and dehydration
reaction catalyzed by human isozyme II as a function of pH by using a
stopped-flow technique. Also by using a fluorescence technique, I have
O i
measured the interaction of Cu with the active site sphere of isozyme
II. The results of these experiments are presented in the second
chapter of my dissertation.
Carbonic anhydrase III is the newest and the slowest isozyme found
in mammals (12,13). (See Appendix A for history, distribution and
physiological function of isozyme III.) Sanyal et aK (14) found that
while cat isozyme III binds anions tighter than isozyme II, it inter
acts 700 to 30,000 times less tightly with the sulfonamide inhibitors.
Tu et al_. (15) showed that the values of the kinetic parameters for the
hydration reaction catalyzed by cat isozyme III are independent of pH
in the pH region 6.0 to 8.5, where in the same pH region, isozyme I and
II have pH dependent activity. Tu et al. (15) also by measuring the
^O-exchange parameters in H2O and D2O found that while the
rate constant k^o has an isotope effect of magnitude 2.5,
exch CO9
k /K c has one of unity. This suggested a rate-contributing proton
cat eff
transfer located outside the interconversion of CO2-HCO3 in
reaction 1 catalyzed by cat isozyme III.


7
All these intriguing findings caused further interest in the
kinetic properties of this isozyme, namely the identification of the
groups which control its activity and binding of inhibitors, and the
elucidation of the rate-contributing proton transfer. An understanding
of these events will then enable us to draw a more complete picture
about the general mechanism of the catalysis by all carbonic anhydrase
isozymes. For this purpose, I have measured several kinetic properties
of cat isozyme III by using stopped-flow, fluorescence, and
^O-exchange techniques. The third chapter of my dissertation
describes the pH profiles of the hydration, dehydration, and esterase
activity of cat isozyme III. In the fourth chapter, the solvent
deuterium isotope and buffer effects on C0£ hydration are presented.
The fifth chapter includes the results of the inhibition by anions and
sulfonamides of the hydration of CO2 catalyzed by cat isozyme III.


CHAPTER TWO
INHIBITION BY CUPRIC IONS OF HYDRATION OF C02
AND DEHYDRATION OF HCO3 CATALYZED BY HUMAN CARBONIC ANHYDRASE II
Introduction
The inhibition by cupric ions of the hydration of CO2 catalyzed by
carbonic anhydrase II is interesting because of the recent results of
Tu et al_. (11) suggesting that Cu2+ is specifically inhibiting a proton
shuttle mechanism in the catalytic pathway. Tu et al_. based this
O 1 O i
conclusion on their observation that Cu as well as Hg^ in the range
of 10"7 to 10" M inhibited the release from the enzyme of H2O bearing
substrate oxygen, but did not affect the actual interconversion of
CO2 and HCO3 at chemical equilibrium. It has been known for some time
that Cu is an inhibitor of the catalysis measured at steady state
pi
(16). The purpose of this study is to examine this inhibition by Cu
at steady state in detail in order to understand more fully the
implication of inhibition by copper of this catalysis. For this
purpose, the inhibition by CuSO^ of hydration of CO2 catalyzed by human
isozyme II was measured, observing the rate of change of a pH-indicator
in a stopped-flow spectrophotometer. These results are used to devise
a model for the inhibition of carbonic anhydrase, a model which is then
supported by simulations of the catalysis (Appendix B). It is the
overall conclusion that copper inhibits intramolecular proton transfer
in carbonic anhydrase II by binding to a residue in the active-site
cleft, probably the imidazole ring of His 64, which participates in the
transfer of protons between the active site and solution.
8


9
Experimental Procedure
Materials. Human carbonic an'nydrase II was obtained from human
erythrocytes of outdated blood. The erythrocytes were washed three
times with isotonic saline and then lysed by addition of a volume of
water equal to the volume of packed cells. The separation of membranes
was achieved by centrifugation and human carbonic annydrase II was
prepared from the supernatant by the affinity chromatography procedure
of Khalifah j?t al_. (17) in the following way. The pH of supernatant
was brought to 8.7 with solid Tris and then gently stirred overnight
with the affinity chromatographic resin containing CM-Bio-Gel A
(Bio-Rad Laboratories, Richmond CA) coupled to p-aminomethyl benzene
sulfonamide (pAMBS). This gel was prepared by the method of Khal ifah
et al_. (17) and washed several times with 0.1 M Tris-sulfate/0.2 M
sodium sulfate, pH 8.7, prior to its use. The following day, the fluid
mixture was passed through a sintered glass filter into a vacuum flaks.
The affinity gel on the filter was thoroughly washed with the same
buffer to remove any nonspecific protein which might adhere to the
affinity gel. To elute the enzyme, 0.1 M Tris-sulfate/0.4 M sodium
azide, pH 7.0 was used. The azide containing protein mixture was
concentrated and then dialyzed for a week at 2C against 9 changes of a
large volume of water.
Enzyme concentrations were estimated from absorbance measurements
at 280 nm by using a molar extinction coefficient of 5.34 x 10^ M-*
cm-* (18). The buffers 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid (Hepes), 4-morpholinepropanesulfonic acid (Mops), and


10
l-4-piperazinebis(ethanesulfonic acid) (Pipes) and indicators
chlorophenol red, bromocresol purple, phenol red, and p-nitrophenol
were obtained from Sigma Chemical Co. 5-Dimethylaminonaphthalene-1-
sulfonamide (DNSA) was synthesized from dansyl chloride by treatment
with ammonium hydroxide by the method of Weber (19) and recrystallized
from ethanol three times. The extinction coefficient of DNSA at 280 nm
O 1 1
was 6.75 x 10J NT1 cm Distilled, deionized water was passed through
an ion-exchange column (Cole-Parmer 1506-35) prior to use to avoid
contamination with adventitious metals. All glassware was soaked in
solutions of EDTA, rinsed thoroughly, and dried before use.
Kinetic methods. Saturated solutions of CO2 were made by bubbling
C02 gas into water in a vessel maintained at 25C. Solutions of CO2
v/ere prepared in the absence of air by diluting saturated solutions
with degassed water using two connected syringes. Concentrations of C02
were calculated based on the concentration of 33.8 mM at 25C for a
saturated solution (20).
Measurements of the initial velocity of hydration of C02 and
dehydration of HCO3 were carried out on a Durrum-Gibson (Model D-110)
stopped-flow spectrophotometer equipped with a Micolet Explorer Model
206 digital oscilloscope. All experiments were performed at 25C. One
drive syringe contained a solution of C02, and the second contained
enzyme, buffer, indicator, and 33 mM Na2S04 (to contribute an ionic
strength of 0.1 after mixing). The initial velocity of hydration (v)
was determined according to Eq. (2), with dA/dt, the rate of change of
absorbance of indicator, obtained by least-squares analysis of


11
absorbance versus time plots using an interfaced Hewlett-Packard 9835 B
cal culator.
. ... , .. f d[CO2]
initial velocity = c
dt
\ / initial
-*00
(2)
initial
Initial velocities were obtained from the first 5 to 10% of the
reaction with each velocity an average of four absorbance versus time
traces.
The buffer factors Q0 relate changes in absorbance to changes in
[H+] and were calculated by the method of Khalifah (21) which uses the
equation
[Buff] K',ff ( K¡nd f [H+]
Qf = X X -
[Ind] LAe Ind \ ,/Buff A [H+]
Ka \K-a +
where [Buff] and [Ind] represent total buffer and indicator
concentrations, respectively, Kau^and K*nd are the buffer and
indicator ionization constants, Ac is the difference in molar extinction
coefficient between the acidic and basic forms of the indicator, and L
is the reaction-cell optical path length in cm. In stopped-flow
studies, the following buffer-indicator pairs were used (also listed
pKa and A£ values): Pipes (pKa = 6.8) with bromocresol purple (pKa =
6.8, X = 588 nm, Ac = 3.00 x 10^ M-* cm-*); Mops (pKa = 7.1) with
p-nitrophenol (p!
Hepes (pKg = 7.5) with phenol red (pKa = 7.5, X = 557 nm, Ae = 5.59 x
10^ M-* cm-*).
For each catalyzed, initial velocity reported, the experimentally
determined uncatalyzed rates have been subtracted. Estimation of
kinetic constants from initial velocities was done using the


12
least-squares method of Wilkinson (22) and Cleland (23) with v^
weights- Such a weight is necessary when the kinetic data are analyzed
in double-reciprocal form of the Michaelis-Menten equation in which the
variance in (1/v) as a function of 1/s is not a constant value.
Fluorescence measurements. Solutions of human carbonic anhydrase
II (4.0 x 10" ^ M) containing various concentrations of CuSO^ and DMSA
were irradiated at 280 nm and the fluorescence emission at 460 nm
caused by the DNSA bound to enzyme was measured (see Chen and Kernohan
[24] for details of the spectra and energy transfer). DNSA is a
sulfonamide inhibitor of carbonic anhydrase. Fluorescence measurements
were made at 25C with a Perkin-Elmer MPF-44B Spectrofluorimeter.
Solutions also contained 50 mM Mops; no Ma2S4 was added (although pH
was adjusted with NaOH and H2S04). Copper was added first at the
desired concentration followed by repeated additions of DMSA. In all
cases, dilution caused by addition of DMSA was less than 2% of the
total volume of enzyme solution (2.5 ml).
O.L
In all experiments, the dissociation constants of Cuc and DMSA
were much greater than the enzyme concentrations; hence, no correction
was made for the amount of inhibitor bound to enzyme. The observed
intensities of fluorescence were corrected for absorption of incident
light at 280 nm by DMSA using the relationship
Fcorr ^obs
2.3e280c
(1 10-£280C)
where e280 1S the mo^ar extinction coefficient of DMSA and C
concentration; Fcorr and F^ are the corrected and observed
(4)
is its
fluorescence intensities. The magnitude of this correction is very


13
small, less than 3% of FQbs in all cases. The fluorescence of unbound
DNSA at 460 nm is negligible (24).
Data are presented (Figure 5) using the Eadie-Hofstee form of the
Langmuir equation based on the intensity of the corrected fluorescence
at 460 nm. The equilibrium dissociation constant of DNSA was estimated
from the Langmuir equation using the nonlinear, least-squares method of
Wilkinson (22) and Cl el and (23) with no weighting factors.
Results
The inhibition by CuS04 of the hydration of C02 catalyzed by human
carbonic anhydrase II at pH 6.0 was noncompetitive (Figure 1); at pH
7.2 it was mixed (Figure 2), and at pH 7.4 it was noncompetitive (data
not shown). Studies at values of pH greater than 7.6 were not
successful because of the complication of formation of hydroxides of
copper. Data such as those in Figures 1 and 2 were fit by
least-squares methods to Equation (5).
1
v
1
Vmax
JiU -i-
KsloPe/ CCO2H
(5)
The inhibition constants Kj^Pe and Kjnt are obtained from the
slopes and the intercepts of double reciprocal plots. Any inhibition
pattern with values of the ratio, Kjn^/KPe, close to 1 and 0 within
experimental errors are called noncompetitive and uncompetitive,
respectively. If this value is very large, approaching infinity,
then the pattern is described as competitive. A mixed inhibition is
the case when the values of this ratio are other than the values
mentioned above. The values of pKjand pKj^^e for inhibition of C02
hydration by Cu^+ are given in Figure 3 (standard derivations for Kjnt


Figure 1. The inhibition by CUSO4 of the hydration of CO2 catalyzed by
human carbonic anhydrase II, present at 1.5 x 10"? M, at pH
6.0 and 25C. Concentrations of CUSO4 were: (o) 0 M, (0)
3.0 x 10-6 M, (a) 4.5 x 105 M, (a) 6.0 x 106 M. Ionic
strength was the sum of that contributed by 33 mM ^$04 and
15 mM Pipes. Lines were determined by weighted least-
squares analysis which resulted in the following constants
C0o fT
(mean and standard deviations): kca^. = (1.7 0.1) x 10
1 CO? int c
sec-1, Km = 7.7 0.5 mM, Kj = (5 0.4) x 10b M,
Kj1ope = (5.4 0.6) x 106 M.


CV1
OJ
I O
o .
2_OlM09S|_W)
.01 X |_w


Figure 2. The inhibition by CUSO4 of the hydration of CO2 catalyzed by
human carbonic anhydrase II, present at 8.0 x 10~8 m, at pH
7.2 and 25C. Concentrations of CUSO4 were: (o) 0 M, (o)
3.0 x IO-7 M, (a) 6.0 x lO-7 M, () 9.0 x 10~7 M. Ionic
strength was the sum of that contributed by 33 mM Ma2S04 and
15 mM Mops. Lines were determined by weighted least squares
COo q 1 COo
analysis and gave: kcat = (3.6 0.1) x 10J sec-1, Km =
i nt si ope
5.7 0.4 nil, Kt = (9 1) x 10~7 M, K¡ = (1.5 0.3) x
10*6 M.




Figure 3. The pH dependence of pKjnt (filled symbols) and pK^or)e
(open symbols) for the inhibition of the hydration of CO2
catalyzed by human carbonic anhydrase II at 25C. Solutions
contained 33 mM Na2S04 and one of the following buffers:
(o) Pipes at 15 mM, () Mops at 15 mM, (a) Hepes at 10 mM.
Bars are standard deviations from least squares analysis.
The line drawn through pK^^e was obtained using equation 6
and gave Kj = (6.7 3) x 10~7, pK^i = 6.9 0.1 and pK^
7.2 0.1.


6.2
7.0
7.8
pH
7.4


and Kj^ope are given for selected points in the Figure). At pH 6.0,
Kjnt was equal to Kj^ope (noncompetitive). The values of Kj were
smallest near pH 7 where inhibition was mixed. At pH near 7.4, the
inhibition was again close to noncompetitive. All of the inhibitory
effects of Cu could be reversed by addition of EDTA. The line drawn
through pKj^ope was obtained by using the equation
slope
Kl
(1 +
[H+j
KE1
kE2>
[H+r
(6)
where Kj is the lowest value of Kj^ope and Kq and are the acid
dissociation constants for the first and second group which control the
binding of Cu^+ (see Figure legend 3 for the values of these
parameters).
Linear Dixon plots were observed for the inhibition of CO2
hydration at pH 7 and of dehydration at pH 6.7 up to concentrations of
1.2 x 10" m Cu^+. No difference in inhibition was observed when Cu^+
was placed either in the drive syringe with enzyme or in the drive
syringe with CO2. Also, I have used buffers of the type studied by
Good et ^1_. (25) that have only weak binding interactions with many
metals (however, see Nakon and Krishnamoorty [26]). In this study, we
have made no correction for the possible binding of Cu to buffer.
O _
The inhibition by Cu of the dehydration of HCO3 catalyzed by
carbonic anhydrase II at pH 6.8 was mixed with Kjn^ = (9 2) x 10"^ M
and Kj^ope = (4 1) x 10" M (data not shown).
O 1
The inhibition caused by Cu at fixed CO2 concentration and varied
buffer concentration gave patterns indicating uncompetitive inhibition
in the pH range 6.8 to 7.4. Figure 4 is a typical result at pH 6.8
using the buffer Pipes.


Figure 4. The inhibition by CUSO4 of the protonation of buffer, B is
Pipes, during the hydration of CO2 catalyzed by human
carbonic anhydrase II, present at 2.5 x 10-8 m at pH 6.8.
CO2 was constant at 5.1 mM and Pipes was varied from 0.35 to
3.0 mM. Concentrations of CUSO4 were: (o) 0 M, (0) 2.0 x
10-7 m, (a) 4.0 x 10_7 Ma2S04 was present at 33 mM.
From least squares analysis, Kjnt = (4.8 1.5) x 107 M.




23
The interaction between Cir2+ and DMSA binding to carbonic anhydrase
II was noncompetitive as determined by fluorescence quenching at pH 7.5
(Figure 5). Cupric ions had no effect on the fluorescence of DMSA in
the absence of carbonic anhydrase, at the concentrations studied here.
From the data of Figure 5, the value of the equilibrium dissociation
constant for Cu2+ is (2.0 0.2) x 10-6 M, and that for DMSA is (1.5
0.1) x 10M at pH 7.5. I have not determined whether Cu^+ and DNSA
are able to bind to carbonic anhydrase II simultaneously or whether
their binding is mutually exclusive. When the binding of DMSA to
bovine red cell carbonic anhydrase II was determined by fluorescence
measurements at pH 7.5 using 20 mM phosphate buffer as solvent, I found
the equilibrium dissociation constant to be 3 x 10"^ M, in agreement
with the results of Chen and Kernohan (24).
Discussion
First, I comment that the range of concentration for the inhibition
o .
by Cir of the hydration of C02 catalyzed by carbonic anhydrase II is
comparable for many different methods of measurement. This study
(Figure 3) has shown strongest inhibition at pH near 6.8 with Kj ~ 8 x
10-'7 M. Magid (16) measured the initial velocity of catalyzed
dehydration of bicarbonate using a pH-titration (pH-stat) procedure at
pH 7.3. He reported 50% inhibition of catalysis near 5 x 10^ M CuSO^
(see Figure 2 of Magid [16]). In the fluorescence quenching
pi
experiments, the maximal binding of Cu occurs above pH 7.3 with
~10"6 m. Tu et jil_. (11) have reported the inhibition by Cu^+ and
Hg^+ of the exchange of ^0 between C02 and H20 catalyzed by human
carbonic anhydrase II. They found that cupric ions and mercuric ions


Figure 5. The percent of maximal corrected fluorescence at 460 nm of
the bound complex of DNSA and human carbonic anhydrase II in
the presence and absence of CUSO4. The concentration of
enzyme was 4.0 x 10? M and Mops was present at 50 mM at pH
7.5 and 25C. Concentrations of CUSO4 were: (o) 0 M, (0)
5.0 x IO-7 M, (a) 1.0 x 10-6 M> () 2.0 x 10-6 M> 4.0 x
106 M. Lines were determined by nonlinear least squares
analysis which resulted in K^TSs for DNSA of 1*5 x 10-6 M.




26
18
inhibit the release from the enzyme of H2 0, but not the
i nterconversion of C02 and HCO3. The data of Tu et jil_. (11) show 50£
inhibition of the release H2*80 at 1 x 10"7 M Cu2+ at pH 7.3. This
value was obtained using solutions containing no buffers and avoided
any competition for copper binding between buffer and enzyme. Hence,
there is reasonable agreement among these various methods in the
concentration of Cu2+ that inhibits the hydration of C02 catalyzed by
isozyme II. It is useful to note that no inhibition of isozyme I is
observed at pH 7 for Cu2+ present in the micromolar range (16,11).
Both the stopped-flow studies (Figure 3) and the results for *80
exchange show maximal inhibition of hydration catalyzed by carbonic
anhydrase II at pH near 7, with inhibition much weaker at either pH 6.0
O .
or 7.6. These observations are consistent with the binding of Cu to
the suggested proton shuttle, the imidazole ring of His 64 which is a
favorable metal interaction site (27). This ring has been shown by NMR
studies to have a pKa of 7.1 (28). Thus, as pH is lowered from 7, this
imidazole ring becomes protonated and loses its capacity to bind copper
tightly. As pH is increased above 7, inhibition by Cu2+ also becomes
weaker. There is no clear explanation of this although Tu et £]_. (11)
suggested that another residue of the active site cleft, not bound by
Cu2+, could act as a proton shuttle at pH > 7. Another possibility is
the formation of hydroxides of copper as pH approaches 8, causing less
copper to be bound to enzyme.
What is clear from the fluorescence studies (Figure 5) is the
pi
noncompetitive interaction between Cu and DNSA in binding to carbonic
anhydrase II. This is quite consistent with the suggestion of Tu et


27
al. (11) that Cu^+ binds to the proton shuttle group, His 64, and
not at the site of binding of sulfonamides, which is the coordination
shell of zinc. Further, Li 1 jas has found, from x-ray diffraction data,
that Hg^+ coordinates to the imidazole ring of His 64 in human enzyme
II.1 In the crystal structure of carbonic anhydrase, the imidazole
o
ring of His 64 is about 6 A from the zinc (8), and sulfonamide nitrogen
binds directly to the zinc (29,30).
Finally, I comment on the mode of inhibition by Cu^+ of catalyzed
hydration. The present understanding of this catalysis by isozyme II
is that both intra- and intermolecular proton transfers can be
rate-limiting (31). This means that the concentrations of the various
enzyme forms (e.g. E, EH+, HEH2+, . ) during catalysis at steady
state can be quite different from the concentrations of these forms
during catalysis under equilibrium conditions (such as ^0 exchange).
Tu et al_. (11) in their equilibrium experiments found evidence that
Cu^+ inhibits the proton shuttle mechanism but not the catalytic
interconversion of CO2 and HCO3. If it is assumed that all forms of
the enzyme are in their equilibrium ionization states, then the result
of Tu et aj_. predicts uncompetitive inhibition of CO2 hydration by Cu^+
at steady state. That this is not observed (Figures 1-3) indicates
that the interpretation of present steady-state data cannot rely on
equilibrium protonation states of all residues involved in the
catalysis. This conclusion has already been reached in the explanation
of the inhibition of CO2 hydration by anions at pH > 8 (7,32). Thus,
Anders Liljas, University of Uppsala, personal communication.


28
to interpret the mode of inhibition of CO2 hydration by Cu^+ requires
O i
that a specific model of the binding of Cu to the enzyme be
constructed and the effect of this binding on catalysis be calculated
by simulation at steady-state conditions. This is done in Appendix B
which confirms that the steady state data reported here are consistent
with the inhibition by Cu of the proton shuttle mechanism.
One aspect of copper inhibition is subject to a simple
interpretation. The mode of inhibition of the protonation of buffer
during the hydration reaction (Figure 4) is uncompetitive. This is
consistent with the suggestion that His 64 is a proton shuttle residue
which must be deprotonated by transfer of H+ to buffer before Cu^+ can
O 1
bind. Thus the binding of Cu depends on the prior interaction of
enzymes with substrate buffer before Cu^+ can bind, consistent with
uncompetitive inhibition.
In summary, these studies confirm the inhibition by cupric ions of
the hydration of CO2 catalyzed by carbonic anhydrase II with a Kj for
binding of Cu^+ near 1 x 10 M. The pH dependence of this binding is
consistent with a binding site on the enzyme with a p'
the imidazole side chain of His 64. This binding site is distinct from
the coordination sphere of zinc in which the actual interconversion of
CO2 and HCO3 and the binding of sulfonamides takes place.


CHAPTER THREE
HYDRATION, DEHYDRATION AND ESTERASE ACTIVITY
OF CAT CARBONIC ANHYDRASE III
Introduction
The results of experiments by Tu et al_. (15) at steady state and
chemical equilibrium which showed pH independent values of the kinetic
CO? CO? exch CO?
parameters, kcat, Km and kcat /Keff for the hydration of CO?
catalyzed by cat carbonic anhydrase III in the pH range 6.0 to 8.5
suggested that the pKa of the activity controlling group(s) in this
isozyme is outside of this pH range. However, Koester jrt jil_. (33)
found that the hydrolysis of p-nitrophenyl acetate catalyzed by isozyme
III from rabbit was dependent on a catalytic group titrating with a pKa
close to 7.5. Although the sequence of neither of the enzymes is
available yet, if one assumes that they are comparable, then the
results of studies by these investigations suggest that CO? hydration
and esterase activities of isozyme III might depend on the ionization
of different active site groups. Since hydration of C0?, dehydration
of HCO3 and hydrolysis of esters catalyzed by carbonic anhydrase II
depend on the ionization of the same catalytic group, zinc-bound H?0
with a p'
control the same reactions catalyzed by cat carbonic anhydrase III.
For this purpose, I have extended the studies of the hydration of
CO? catalyzed by cat carbonic anhydrase III to a wider region of pH and
29


30
measured dehydration of HCO3 and hydrolysis of p-m'trophenyT acetate
catalyzed by cat isozyme III in the pH region 5.7 to 9.7. The kinetics
of catalysis of CO2 hydration and HCO3 dehydration were observed by a
stopped-flow method (21) in which velocities were determined from the
rate of change in absorbance of a pH indicator. The kinetics of the
hydrolysis of p-nitrophenyl acetate were determined by following the
appearance of the product p-nitrophenoxide at 400 nm using a
spectrophotometer.
I found that the hydration, dehydration and esterase pH-rate
profiles of cat isozyme III are consistent with this enzyme having an
activity controlling group with a pKg < 6. The results also indicate
an additional active site group with a pKg ~9.0, the ionization of
which increases the catalytic activity of isozyme III above pH 8.
Experimental Procedure
Methods. Carbonic anhydrase III was prepared from the hind limbs
of fully anesthetized cats (pentobarbital, 25 mg/kg, i.v.). After
administration of heparin (3000 units/kd, i.v.), the hind limb was
perfused with 0.9% NaCl until the muscles appeared pale and the venous
effluent was colorless. The muscle was excised, homogenized, and the
homogenate centrifuged. Carbonic anhydrase III was isolated from the
supernatant by the method of Sanya! et jil_. (14) which uses two affinity
gels: first with p-amninomethyl benzene sulfonamide coupled to
carboxymethyl (CM)-Bio-Gel A (Bio-Rad) to remove erythrocyte carbonic
anhydrase I and II, and second with 2-(4-aminobenzene)sulfonamide-
l,3,4-thiadiazole-5-sul fonamide coupled to (CM)-Bio-Gel A to isolate
carbonic anhdyrase III. The second gel was then rinsed clean with Tris


31
sulfate buffer at pH 7.5, following which carbonic anhydrase III was
recovered by elution with 0.4 M sodium azide. This solution was
dialyzed against several changes of a large volume of 1 mM
dithiothreitol (DTT) solution. To separate the monomer from the dimer,
the purified enzyme was passed through an Ultra Gel (LKB AcA 44)
molecular sieve column pre-equilibrated with 0.05 M Tris sulfate,
0.025 M Na^O^, and 1 mM dithiothreitol at pH 7.5. The monomeric
enzyme fraction was collected and dialyzed against 1 mM dithiothreitol
and stored at 4C in the same reducing medium. Only the monomeric form
of cat isozyme III was used in these studies. The purity of the sample
was estimated by polyacrylamide gel electrophoresis and with the
inhibitor ethoxzolamide, which is a potent inhibitor of human isozymes
I and II (Kj = 2 x 10"^ M) and a weak inhibitor of cat isozyme III
(Kj= 5 x 10-5 M) (measured at 0C, see reference 14). By neither of
these methods, no contamination by isozyme I or II was detected. The
concentration of cat isozyme III was determined from the molar
extinction coefficient of 4.8 x 10^ cm"-*- (14).
Kinetic method. Solutions of C02 were made up in the same way as
described in Chapter Two. For the dehydration reaction, appropriate
concentrations of KHCO3 were prepared by diluting freshly prepared
0.2 M stock solutions of KHCO3 with C02-free H20. All the experiments
were performed at 25C. Moreover, all solutions contained 33 mM Na2S04
to give an ionic strength of 0.1.
Initial velocity measurements of C02 hydration and HCO3 dehydration
were carried out on a Durrum-Gibson stopped-flow spectrophotometer in
the same v/ay as described in Chapter Two. In stopped-flow studies, the


32
following buffer-indicator pairs viere used (also given are pKa values,
wavelengths used and differences in molar extinction coefficients of
acidic and basic forms): 4-morpholineethanesulfonic acid (Mes) (pKg =
6.1) and 3,5-lutidine (pKa = 6.2) with chlorophenol red (pKa = 6.3, X =
574 nm, Ac = 1.77 x 10^ M* cm"-'-); l,4-piperazinebis(ethanesulfonic
acid) (Pipes) (pKa = 6.8) with bromocresol purple (pKa = 6.8, X =
588 nM, Ac = 3.0 x 10^ M-* cm"-*-); 4-morpholinepropanesulfonic acid
(Mops) (pKa = 7.1) and 1-methylimidazol (pKg = 7.1) with p-nitrophenol
(pKg = 7.1, X = 400 nm, Ac = 1.83 x 10^ M-* cm-*); 4-(2-hydroxethyl)-l-
piperazineethanesulfonic acid (Hepes) (pKa = 7.5) with phenol red
(pKa = 7.5, X = 557 nm, Ac = 5.59 x 10^ M-* cm-*); 1,2-dimethyl imidazole
(pKa = 8.2) with m-cresol purple (pKg = 8.3, X = 578 nM, Ae = 3.81 x
10^ M-* cm-*); l,4-diazabicyclo-[2.2.2]octane (Ted) (pKg = 9.0) with
thymol blue (pKa = 8.9, X = 590 nM, Ac = 2.43 x 10^ M-* cm-*).
Measurements of esterase activity were performed at room
temperature similar to previously described procedures (34,35) using
p-nitrophenyl acetate as substrate. The hydrolysis of p-nitrophenyl
acetate was followed by measuring the appearance of p-nitrop'nenoxide
and p-nitrophenol at 400 nM by using a Beckman spectrophotometer Model
UV 5260. At this wavelength, e = 1.83 x 10^ M-* cm-* for
p-nitrophenoxide and e = 200 M-* cm-* for p-nitrophenol were used (34).
A pKa of 7.14 for p-nitrophenol was assumed (36).
The following procedure was used for activity measurements. A
fresh 40 nil solution of p-nitrophenol acetate was prepared in 10 ml
acetone and was kept in ice during the experiment. First, enzyme v/as
mixed with 50 mM buffer/33 mM f^SO^ solution in a 1.5 ml volume


33
cuvette. Then to that 50 pi of substrate solution was added to give
2 mM final concentration in 1 ml total reaction volume. This procedure
was repeated at 5 different enzyme concentrations from 0.6 x 10^ to
2.2 x 10"^ M at each pH, also the uncatalyzed rates were determined
under the same conditions without enzyme and subtracted from the
catalyzed rates. Data were plotted as the rates on the Y and enzyme
concentrations on the X coordinate and the slopes of these lines which
are kenz [S] were obtained by using a least squares method (22,23).
kenz is equivalent to kcat/Km only when the concentration of substrate
is lower than the values of Km.
Results
C02
Hydration activity. The turnover number kca^ and Michaelis-Menten
C02
constant Km for the hydration of C02 catalyzed by cat carbonic
anhydrase III were independent of pH in the pH range from 5.7 to 8.0 as
was also observed by Tu et al_. (15). When pH was increased above 8,
C02 C02
both kcaj. and Km increased and did not reach plateau at pH 9.6
, C02 CO?
(Figure 6, open symbols). Although the increases in kcat/Km above pH
C02
8 were not as large as the increase in kcaj., at pH 9.6 the value of
C02 CO?
kcat/Km was more t*ian doubled with reference to its value in the pH
region 5.7 to 8.0 (Figure 6). In Table 2, the average values of these
kinetic parameters at the indicated pH values are given. The line
drav/n through the experimental points in Figure 6 was obtained by using
the equation
"cat
= ) x
cati kE17 [H+]
(kc2 )
+ (kcat2}
KE1 (kcat?)D .. KE2
Ke2 + [H+]
(7)


Figure 6
C0?
The upper panel indicates the pH dependence of log kca£,
logarithm of the turnover number of the hydration of CO2
catalyzed by carbonic anhydrase III from cat muscle in H2O
(open symbols) and 98% D2O (closed symbols). Temperature
was 25C and solutions contained 33 mM of Na2S4 and the
concentration of the following buffers: ( 3,5-lutidine; (O) 10 mM 1-methylimidzaole; (o) 20 mM Mes;
(0) 15 mM Mops; (m) 20 mM Hepes; (v) 20 mM 1,2-
dimethyl imidazole; (a) 20 mM Ted. The line drawn through
the experimental points were obtained using equation 6 with
the values of (kcati) = 1*8 x 10^ sec_l; (k^a^)0 = 1*4 x
10^ sec~l; pKci = 5.3 and pKp2 = 9.1. The lower panel
C02 C02
indicates the pH dependence of kca|/Km for the hydration
of CO2 catalyzed by carbonic anhydrase III from cat muscle
measured in H2O (open symbols) and 98% D2O (closed symbols),
Symbols and conditions are the same as in the upper panel.


pH


36
TABLE 2
THE VALUES OF THE KINETIC PARAMETERS FOR THE HYDRATION,
DEHYDRATION AND p-NITRO PHENYLACETATE ESTERASE ACTIVITY
OF CARBONIC ANHYDRASE III AT THE INDICATED pH VALUES
Parameter Value at Low pH Value at High pH Dimension
C02
^cat
(cat)
^2>
(cat)
cC02/C02
^cat^m
(cat)
d COp
4cat (bovine)
d CO9
(bovine)
(1.8 0.5) x 103
12 5
(1.4 0.5) x 105
(1.2 0.1) x 103
9 1
0HCO3 HCO3
Aat /Km (cat)
4
enz
(cat)
4
enz
(rabbit)
7.5 x 104
0.6 0.2
1.2
(1.0 0.3) x 104
( 30 10 )
( 3 0.3) x 105
(1.2 0.2) x 104
26 6
4.6 x 103
4.5 0.8
5.2
sec
rrfl
M-* sec-*
sec-*
mM
M-* sec-*
M-* sec-*
M-* sec-*
2 k r
.\/aiues at low pH and high pH were calculated by taking the
average values of the kinetic parameters in the pH region from 5.7 to
7.8, and at pH 9.5 and presented with their standard deviations.
4)ata at pH 7.4 (in 30 mM Hepes) and pH 9.3 (in 30 mM Ted).
4ow pH value is 6.0 and high pH value is 7.4.
4pnz (esterase reaction) from this study: low pH = 6.7; high
pH = 8.7.
k from Koester et al. (33): low pH = 6.7; high pH = 8.7.
1
enz


37
pn pA aa
where (kca£) and (kca^2) are the maximum values of kca£ in the pH
regions 6.0 to 8.0 and greater than 9.6, KE1 and KE2 are the acid
dissociation constants of the first and second enzymic group which
control the hydration activity of isozyme III (see Figure 6 for the
values of these parameters). A result similar to that given in figure
6 (open symbols) was also observed by using bovine carbonic anhydrase
COp COp
III. The values of kcat and Km at the indicated pH values are given
in Table 2.
HCO3 HCO3
Dehydration activity. The pH dependence of kcat /Km for the
dehydration of HCO3 catalyzed by cat isozyme III is presented in Figure
7. Because of the difficulties in getting sufficiently accurate data
due to the uncatalyzed rates being as high as 25-40% of the catalyzed
HC03
rates, and also high values of Km with high uncertainties
especially at increasing pH values, no attempt was made to interpret
HCO3 HCO3
kcat anc* ^m values individually. The data shown in Figure 7 are in
approximate accordance with the following expression which describes
the observed increase in activity when pH is lowered:
HCO3
ccat
,hco5
HC05
vcat
,hco5
[H+]
(8)
Km Km [H+]+Ke1
where the subscript 0 reflects to a maximum value (see Figure legend 7
for the values of parameters used in this fitting). In Table 2, the
HCO3 HCO3
values of kca£ /Km at low and high pH values are given.
The observed Michaelis-Menten parameters given in Figures 6 and 7
can be related through Haldane relationship (equation 9) to the
equilibrium constant of C02 and HCO^ (equation 10).


HCCh HCO3
Figure 7. Logarithm of kca^ /Km for the dehydration of HCO3
catalyzed by cat carbonic anhydrase III versus pH, at 25C.
Ionic strength was determined by the presence of 33 mM
Na2S04 and 20 mM of the following buffers: (<) 3,5-
lutidine, (O) Pipes, (O) Mops and () Hepes. The line
drawn through experimental points was obtained using
equation 7 with the following values of parameters:
(kcat^/KmCV = 6.0 x 105 M"1 sec"1, pKE1 = 5.2.


6.0 7.0 8.0
6£


-tu
CO? HCO3
t, 1/ J y
Kcat x Nin = Neq
K
co2 hco3 [h+]
(9)
m
xcat
where is
[HCO3XH ]
e9 [C02]
(10)
Insertion of the data of Figure 6 and 7 in equation 9 yields K
eq
1.7 x 10"^ M which is comparable to the literature value 9.0 x 10"^ M
(37).
Esterase activity. The pH profile of the hydrolysis of
p-nitrophenyl acetate catalyzed by cat isozyme III is shown in Figure
8. Because of the limited solubility of p-nitrophenyl acetate, kenz was
measured. As can be seen, the shape of Figure 8 looks similar to that
of Figure 6 in that kgnz is also independent of pH in the pH range 6.2
to 7.6 and increases above pH 7.6. The line drawn through the
experimental points in Figure 8 was obtained using equation 7 by taking
the same values of Kq and K¡:2 listed in the legend to Figure 6 for C02
hydration. The values of kenz at the indicated pH values are given in
Table 2 together with the values obtained by Koester et al_. (33) using
rabbit isozyme III.
Discussion
C02 C02
The observation of a plateau for kcat and K(n in the pH region 6.0
to 8.0 is the same as Tu et_ aj_. have found (15). This plateau of
activity could very well be the high pH arm of a pH-rate profile having
an activity controlling group with a pKfl lower than 6.0. The
dehydration pH profile (Figure 7) certainly indicates such a low pKa.


Figure 0. Logarithm of the second order rate constant kenz for the
hydrolysis of p-nitrophenyl acetate catalyzed by cat
carbonic anhydrase III versus pH at 25C. p-Nitrophenol
acetate concentration was 2.0 x 10 3 M. Ionic strength was
determined by the presence of 33 mM Na2S04 and 50 mM of the
following buffers: (a) Pipes and (o) Tris-S04. The line
drawn through the experimental points was obtained using
equation 7 with the following values of the parameters:
(kcltl) = 0-6 M'1 sec'1, (kecl\2)0 = 16 M_1 sec'1, pKE1 =
5.3, pKE2 = 9.0.


log kENZ
p o
o CJi
Z\/


43
So it appears that in the pH region 6 to 8, CO2 hydration activity of
cat isozyme III depends on the basic and the dehydration activity
depends on the acidic form of this catalytic group with pKa lower than
6.0. Therefore, if the velocities of the hydration reaction catalyzed
by isozyme III are measured above pH 6.0, only the high plateau region
of pH profile would be seen (Figure 6); where, in the same pH region,
the dehydration pH-rate profile would be pH dependent with increasing
activities as the pH decreases (Figure 7). This result is similar to
what is observed for isozyme II, except its activity controlling group
has a pKg of 7.0.
I suggest that this ionizable group with a pKa lower than 6.0 is
zinc-bound H2O. Further supportive evidence for this hypothesis will
be presented in the following Chapters from the anion inhibition and
solvent deuterium isotope effect studies. Moreover, Engberg and
Lindskog (38) have found that Co(ID-substituted bovine carbonic
anhydrase III has a visible spectrum at pH > 6.0 very similar to that
CO o
of Co(ID-substituted isozyme II at pH > 8. The pH profiles of kca£ for
isozyme III from bovine and cat look similar, which suggests that the
active site regions of both enzymes are comparable. This low pKg for
H2O in isozyme III is perhaps a result of the positively charged
environment created by lysine 64 and arginine 91 which are found in the
active site cleft for bovine isozyme III (39), which could stabilize a
CO? CO?
zinc bound hydroxide. The cause of the increase in kca£ and ' > 8 could be due to the ionization of these basic amino acid residues,
especially lys 64 which would become a proton shuttle group as pH is
increased, and enhance activity by providing a more efficient pathway


44
for transfer of protons from the active site to solution. In this
respect, the function of lys 64 in isozyme III at pH 9 resembles that
of His 64 in isozyme II at pH 7 (1). It is also possible that this
C02
increase in k^ could be caused by an effect of hydroxide ion in
solution acting as a proton acceptor in this catalysis that produces a
proton in addition to HCOj. A turnover number lCp sec-* is consistent
with the maximal rate of proton transfer from a group of pKa around 5
(zinc bound water in isozyme III) to H20 in solution. Hydroxide ion in
solution, being a much better proton acceptor, would be expected to
C02
increase the rate at alkaline pH. The appearance of pH dependent kca|
C02 C02
and pH independent kcat/Km above pH 8 suggests that the change in
... C02
ionization state observed for kcat occurs in an enzyme substrate
complex not in the free enzyme (40).
As shown in Table 2, the values of kenz obtained in this study by
using cat isozyme III agree with the corresponding values obtained by
Koester et al_. (33) using rabbit isozyme III at the indicated pH
values. But, in contrast to their result which showed a pKa 7.5 for
the activity controlling group, the present study shows that the
hydrolysis of p-nitrophenyl acetate catalyzed by cat isozyme III depends
, ... C02
on tne ionizations of the same catalytic groups which effect kca£
(Figure 6) with p'Ka values lower than 6.0 and 9.0.
In summary, the results presented in this chapter are consistent
with carbonic anhydrase III from cat having at least two active site
groups with pa values lower than 6.0 and around 9.0. I suggest that
the catalytic group with the low pKa is zinc-bound H20.


CHAPTER FOUR
RATE-CONTRIBUTING EVENTS IN THE HYDRATION OF
C02 CATALYZED BY CARBONIC ANHYDRASE III
FROM CAT SKELETAL MUSCLE
Introduction
In a recent study, Tu^t jiT_. (15) by using ^0 exchange technique
showed that the magnitudes of the solvent hydrogen isotope effects on
exch COo
kinetic parameters kcat /Keff and k^o (see Chapter One for their
meaning) obtained from the hydration of C0£ catalyzed by cat isozyme
III were unity and 2.5, respectively. This result suggested that the
rate-contributing event in hydration of CO2 catalyzed by cat carbonic
anhydrase III could also be a proton transfer event located outside the
interconversion of C02 and HCO3 as observed for isozyme II (1,2).
exch C02 COo COo
Since kcat /Keff and k^Q are described to be equivalent to kca|/Km
C02 4
and k_a1. at steady state (9), such a result requires that the
C02 C02 C02
magnitudes of isotope effects on kca|/Km and kca^ measured at steady
state should be close to those obtained in ^0 exchange experiments.
The effect of buffers on the release of product proton in the
hydration reaction catalyzed by isozyme II was discussed in Chapter
One. It was not known until this work what effect buffers have on
hydration of CO? catalyzed by isozyme III.
In order to understand the rate-contributing events, the rates of
CO2 hydration catalyzed by cat carbonic anhydrase III were measured in
H2O and 98% D2O in the pH interval from 6.2 to 9.5 by using
45


46
stopped-flow technique. The effect of buffers on the hydration
reaction was studied by using stopped-flow and 180 exchange techniques.
The results of my experiments support the hypothesis of Tu et al_. (15)
that the rate determining event in the catalysis of the hydration
reaction by cat isozyme III is a proton transfer located outside the
interconversion of CO2 and HCO3. The rate of the release of product
proton to solution is not rate limiting at any concentrations of
buffers.
Experimental Procedure
The materials and kinetic methods were similar to that described in
Chapter Three. The concentration of saturated solutions of CO2 in D2O
was taken as 3.8 x 10"^ M (20).
The values of pH(D) reported here are uncorrected pH meter
readings. The correction of a pH meter reading in 100% D2O [pD =
(meter reading) + 0.4] (41) is approximately offset by the change in
the ionization constant of buffer in D2O (pKp pKH = 0.5 0.1) (42).
Consequently, one expects that all reactive species maintain
approximately the same ratio of acidic to basic forms in two
experiments, one in H2O and one in D20 at the same value of the pH
meter reading. The magnitudes of solvent deuterium isotope effects on
kinetic parameters were determined by simply dividing the value of the
parameter in H2O by that in D2O.
1
Equilibrium iO0-exchange experiments were performed as described by
Silverman ert cil_. (9).
Results
CO2 CO? CO?
The values of kcat and kca£/Km obtained in H20 and 98% D20 in the
pH range from 6.0 to 9.5 are presented in Figure 6. In Figure 9, one


Figure 9
Double reciprocal plot showing the catalysis by carbonic
anhydrase III (2 x 10"6 M) from cat muscle of the hydration
of CO2 in H2O (o) and 98% D2O (0) at pH 6.6 and 25C. Na2S04
was present at 33 mM and buffer Mops was used at 15 mM
concentration in both H2O and D2O. Lines were determined by
weighted least-squares analysis (see text) which resulted in
the following constants (mean and standard deviation):
h2, C02v .-5 1 M, COo,
d2, C02t o DoO C02%
(kca|) = 9.1 x 102 sec-1; (Km ) = 8
D# C02,
1 mM; (kcat) =
0, C02l
2.3; (Km ) = 1.75




49
of those hydration experiments performed at pH = 6.6 in HpO and 98% D2O
is given in the double reciprocal form. The average values of the
COp Cop COp Cop
solvent hydrogen isotope effects on kca!j:, Km and kca|/Km obtained
from the data in the pH range 6.2 to 9.5 are tabulated in Table 3. The
COp
ionizations which determine the behavior of kcat for cat isozyme III
are consistent with a change of pKa of (pKj (pKj = 0.5,
a p a p a d2o p a H20
which is the value commonly observed for weak acids with values of pKa
in the range of 5 to 10 and close to that for the ionization constant
for dissociation of HpO (0.6) (43).
By using the stopped-flow technique, it was observed that
concentrations of 2 to 50 mM of Mops at pH 7.1, Hepes at pH 7.5, and
Ted at pH 9.0 caused no change in the rate of hydration of CO2
catalyzed by cat isozyme III. In agreement with this, there was no
change in the catalyzed ^O-exchange rates in the presence of up to
1 O
50 mM mops and hepes compared with the iO0-exchange in the absence of
buffers at pH 7.1.
In contrast to the buffers mentioned above, imidazole caused an
increase in the rates of COp hydration studied by both methods. In the
1 ft
iO0-exchange experiments, the effect of imidazole was mainly on k^Q,
1 ft
the rate constant corresponding to the release of AO0-labeled HpO from
the active site (Figure 10). The values of k-^g in the presence of
30 nil imidazole were almost doubled with reference to its value in the
absence of imidazole at pH 7.1. The value of the other ^O-exchange
exch COp
parameter kcat Aeff showed a consistent 15 to 25% drop at all
concentrations of imidazole with reference to its value in the absence
of imidazole (data not shown). The effect of imidazole on hydration of


50
TABLE 3
SOLVENT HYDROGEN ISOTOPE EFFECTS FOR STEADY-STATE AND
EQUILIBRIUM EXCHANGE CONSTANTS IN THE CATALYSIS OF C02 HYDRATION
D C02a
kcat
D, C02 Ob
^cat^m )
D, exch COp,
(kcat ^eff^
D
kH20
Human
Isozyme II
-O
00

CO
1.0b
1.0 0.1c
4.0 0.5C
Cat
Isozyme III
2.6 0.3d
1.3 0.2d
1.0 0.1e
2.4 0.4e
a. The superscript D indicates solvent hydrogen isotope effect. Thus,
D COp COp COp
kcat (kcat ^O^cat ^O*
b. Data from the high pH plateau (pH > 8) of Steiner et a]_. (1).
c. Data at uncorrected pH meter reading 8.0 from reference 10.
d. The average values of solvent hydrogen isotope effects obtained
from Figure 6 (this work) presented with their corresponding
standard deviations.
e. Data at uncorrected pH meter reading 8.0 from reference 15.


Figure 10. The rate constant k^o (the rate at which water containing *80 label
is released from carbonic anhydrase) vs_ the concentration of
imidazole at pH 7.1 and 25C. The concentration of cat carbonic
anhdrase III was 1.0 x 10~7 M. The ionic strength of solution was
kept constant at each imidazole concentration at 0.2 with ^SO^
Total substrate concentration was 15 mM (CO2 + HCO3).


O 20 40 60
IMIDAZOLE (mM)


53
C02 catalyzed by cat carbonic anhydrase III at steady state was similar
C02 C02
to that shown in Figure 10. The values of kcat and Km obtained in
20 mM imidazole at pH 7.1 were 1.5 x 10^ sec-* and 50 mM, respectively.
These values are 5 to 6 times higher than the values listed in Table 2
using other buffers. By using fluorescence, a quenching method which
will be described in Chapter Five in detail, imidazole, up to 70 mM
concentrations at pH 7.1, did not affect the fluorescence of the
chiorzolamide-cat carbonic anhydrase III complex at 330 nM. Similar
to this, imidazole up to 0.2 M concentrations at pH 9.0 did not affect
the fluorescence of DNSA-human isozyme II complex at 460 mM. However,
the fluorescence of DMSA-human isozyme I complex at pH 9.0 was
decreased by imidazole with a value of IC5Q ~50 mM.
Discussion
There is a very large difference in maximal turnover for hydration
of C02 between isozymes II and III at high pH with a difference of
C02 C02
similar magnitude between kcat/ I would like to point out similarities in the kinetic features of these
isozymes II and III which strongly suggest that they have in common a
rate-contributing event occurring outside the steps involved in the
actual interconversion of C02 and HCO3. This event is suggested to be
a proton transfer for isozyme II and may be a proton transfer or
conformational change for isozyme III (1,2). These conclusions are
drawn from two types of experiments: 1) a comparison of equilibrium
1 ft
lo0-exchange data with steady-state results and 2) solvent hydrogen
isotope effects on the ^O-exchange and steady-state constants.


54
Comparison of ^0 with steady state. Table 4 compares two
constants obtained by equilibrium ^0 exchange with the steady-state
HCO3 exch
turnover number for dehydration, kcat The rate constant kca^. which
was obtained from the rate of exchange of ^0 between CO2 and H20 (9),
13
and has also been determined from the exchange broadening of the C
13 13
resonances of H CO3 and C02 (44), is the maximal rate constant for
interconversion of C02 and HCO3 at chemical equilibrium. Table 4 shows
that this rate constant is greater than either the steady-state
HCO3
turnover number for dehydration kcat or k^o* (The rate constant k^Q
measures the release from the enzyme of oxygen abstracted from
substrate bicarbonate [9].) This implies that the turnover rate at
steady state is limited by a step outside those involved in the
interconversion of C09 and HCOo. The constants (k^?^) and
0 cat max
(kH20*max are aPProxl'mately equal, suggesting that both are determined
by this rate-limiting step. For isozyme II, Simonsson £t aK (44)
noted that such data are consistent with an intramolecular proton
transfer between zinc-bound water and an ionizable group in the
active-site cleft. That this step involved proton transfer was
indicated from the solvent hydrogen isotope effects.
Solvent hydrogen isotope effects. For the hydration of C02
catalyzed by human carbonic anhydrase II and bovine carbonic anhydrase
II, Steiner jrt jiK (1) and Pocker and Bjorkquist (2) showed that the
COo D COo
solvent hydrogen isotope effect of kca£ written kca£, was close to 3.8
for the high pH arm of hydration (pH > 8). However, there was a
C02 C02
solvent hydrogen isotope effect of unity for kcaj./Km The ratio
C02 C02
^cat^m contains rate constants for steps up through the first


55
TABLE 4
MAXIMAL VALUES FOR SOME STEADY STATE AND EQUILIBRIUM
EXCHANGE CONSTANTS IN THE CATALYSIS OF C02 HYDRATION3
, ex ,
(kcat)
cat max
(xlO5)s_1
^HoO^
max
(xlO5)s_1
, hco:
(kcat3)
caT- max
(xl0~5)s_1
Human isozyme II
18
10
6
Human isozyme I
0.4b
0.3
0

GT
Cat isozyme III
>0.1
0.02
0.02
a. is independent of pH for isozymes I and II in the range of
pH 6 to 8 (44,45) The value of kat for isozyme III was determined at
pH 6.6 and 7.1. Maximal values of k^g occur near pH 6.5 for isozymes
II (9) and I. k^g is independent of pH between 6 and 8 for isozyme
HCO3
III. kcat has a maximum near pH 6 for isozyme II and I. For isozyme
HCO3
III the value of kca^. was obtained at pH 6.0.
b. From reference 45.


56
irreversible step which, in the initial velocity experiments, is the
D, C02 C02,
release of HCO3 from the enzyme. The value of unity for 'kcar >
for isozyme II strongly suggests no rate-contributing proton transfer
in the steps involved in the interconversion of C02 and HCO3. The
D exch COo
exchange experiments confirm this in that (kr,t /Kpff) is also unity
, exch C02 C02 C02
(the ratio kcat /Keff is equivalent to kcat/Km ). However, the value
of kca£ of 3.8 indicates a primary proton transfer somewhere in the
catalysis. The exchange experiments reveal a large solvent
hydrogen isotope effect on k^g for isozyme II, as great as 8 at pH
6.6, indicating that the rate-limiting proton transfer is also involved
1 ft
in the steps that release iO0 from the enzyme.
There is a rather close parallel between the results described here
for isozyme II (at pH > 8) and those for cat isozyme III as shown in
D C02 C02l D exch C02l D C02
Table 3: (kCarKm and ^kcat /Kef-r are close to unity> kcat and
D C02
kH20 are very siQO'if'icantly different from unity. We therefore
suggest, in anology with isozyme II, that the hydration of 083
catalyzed by cat isozyme III is limited in turnover rate by a step
occurring outside the interconversion of CO2 and HCO3 and that this
step involves the changing of bonding to hydrogen.
There is no evidence to suggest the nature of the rate-limiting
event for hydration of C02 catalyzed by cat isozyme III. A turnover
number of 104 sec-1 at pH 9 seems a bit slow to be determined in rate
by an intramolecular proton transfer, although this possibility cannot
be eliminated (31). It is part of the hypothesis of Steiner et al_. for
isozyme II that buffers are not able to transfer a proton directly with
the active site group but require an intermediate proton shuttle group


57
(1). One possibility for isozyme III is that there is no suitable
residue near the active site which can act as a proton transfer group
between the active site and buffers in solution. Then, isozyme III
would have to transfer a proton to water at the slow rate of 10^ to 10^
sec-* which can very well be the rate-limiting step in the overall
hydration reaction in H20 and D20. This would also produce no buffer
effect in the catalyzed hydration reaction.
The effect of imidazole on the hydration of C02 catalyzed by cat
isozyme III is not clear yet. Tu ^t al_. (46) also observed that the
phosphate buffer at 25 to 50 mM concentrations caused 100% increase in
1 O
the iO0 exchange parameter k^o obtained using cat isozyme III. It is
therefore possible to suggest that buffers such as imidazole and
phosphate which have relatively smaller sizes than buffers Mops, Hepes
and Ted can get into the active site and by accepting the proton from
zinc-bound H20 can increase the rate of the hydration reaction. The
rates of hydration of C02 obtained by using isozyme II are also
several-fold faster in imidazole buffer (47). Rowlett and Silverman
(32) suggested that this could be due to the ability of imidazole to
get into the active site of isozyme II and short-circuit the proton
shuttling step. Imidazole is a specific inhibitor of C02 hydration
catalyzed by isozyme I with a Kj of 18 mM (21) and is shown to have a
specific binding site within the active site (8). It is apparent from
the fluorescent experiments that the effect of imidazole on the
hydration of C02 catalyzed by isozymes II and III is not due to its
binding anywhere in the active site region of these isozymes. However,
the decrease caused by imidazole in the fluorescence of isozyme I-DNSA


58
complex suggests that inhibition of isozyme I is due to the binding of
imidazole to a specific site in the active site region, consistent with
x-ray data (8).
In summary, carbonic anhydrase III from cat has a rate-limiting
proton transfer which comes after the interconversion of C02 and HCO3
as found for isozyme II. However, in contrast to isozyme II, isozyme
III is not enhanced by buffer in its catalysis.


CHAPTER FIVE
INTERACTION OF CAT CARBONIC ANHYDRASE III
WITH ANIONS AND SULFONAMIDES
Introduction
Anions and sulfonamides are inhibitors of carbonic anhydrase
isozymes which have been shown to coordinate to the active site metal
ion, zinc (8,48,49). In the past, they have been very useful to
elucidate the kinetic mechanisms of isozyme I and II. Anions were
shown to inhibit C02 hydration catalyzed by human carbonic anhydrase I
in a noncompetitive fashion at all pH values (50,51). The
corresponding pattern for carbonic anhydrase II was noncompetitive at
low pH values but was changed to uncompetitive at pH > 8 (50,52). This
difference in the inhibition patterns is perhaps the reflection of the
different rate limiting steps in the kinetic mechanisms of isozymes I
and II. An uncompetitive type of inhibition for isoyzme II was
explained to be consistent with the enzymic model given in Scheme 1
(7,32).
Sulfonamides are potent inhibitors of isozymes I and II (53).
Therefore they equilibrate rather slowly with the enzyme while the
catalysis precedes and at steady state behave as pseudo-irreversible
inhibitors and give rise to noncompetitive patterns with respect to
C02. But a competitive behavior with respect to HCO3 has been reported
(54).
Sanya! _et al_. (14) previously reported that the IC^q values of many
anions obtained from the inhibition of the hydration of C02 catalyzed
59


60
by cat isozyme III were lower than the corresponding numbers obtained
for isozyme II. In their study, they have also shown that the IC^g
values of many sulfonamides, such as ethoxzolamide and acetazolamide
for cat isozyme III, were much higher than the corresponding number for
isozyme I and II (IC^q value of acetazolamide for isozyme II is 10^ M
and for isozyme III is 3.0 x 10'^ M [14]. These results suggested that
the active site of cat carbonic anhydrase III must be quite different
than the active sites of the other isozymes.
To understand further the kinetic mechanism of cat isozyme III and
also the residues which control the binding of anions and sulfonamides,
the inhibition by these inhibitors of the hydration of C0£ catalyzed
by cat isozyme III was measured and the pattern of inhibition and
magnitude of Kj as a function of pH was determined by using a
stopped-flow technique. Direct interaction of anions and sulfonamides
with the active site of cat isozyme III was investigated by using a
fluorescence method.
The results of experiments further support the hypothesis that the
pKa of the catalytic group in cat isozyme III, most likely a zinc bound
water, is lower than 6.0 and that the catalysis by this enzyme is
limited in rate by a step occurring outside the interconversion of CO2
and HCO3. The results also show that another active site residue with
pKa higher than 8 affects the kinetics of inhibition by anions.
Experimental Procedure
Materials. Chlorzolamide (5-[o-chlorophenyl]-l,3,4-thiadiazole-
2-sulfonamide) was obtained from Lederle Laboratories (Pearl River,
NY). Its molar extinction coefficient was determined to be 1.5 x 10^


61
M-1 cm-1 at 280 nm and 1.0 x 10^ cm--1- at 330 nm. Ethoxzolamide was
obtained from Upjohn Drug Company. The salts, NaNg and KI, were
obtained from Aldrich Chemical Company. The rest of the materials were
the same as described in Chapter Four.
In inhibition experiments, Nj and I" were chosen as inhibitors of
the hydration of CO2 catalyzed by cat isozyme III because of their IC^q
values which were reported to be relatively higher than some of the
other anions such as CN" and OCN- (14) and also higher than the enzyme
concentrations employed in these experiments ("2.0 x 10-^ M). This
precaution was taken to avoid any tight binding by those inhibitors
which will equilibrate with the enzyme slowly while the catalysis is
proceeding at steady state and therefore would effect the results of
inhibition experiments. In stopped-flow procedures, these inhibitors,
including sulfonamides, were mixed with the solution containing enzyme,
buffer and indicator prior to the mixing with substrate. When the
inhibitor iodide was mixed with substrate solution prior to the mixing
with the enzyme, there was no difference in inhibition results.
Fluorescence Quenching. We observed the quenching of the
tryptophan fluorescence of cat carbonic anhydrase III caused by the
binding of the inhibitor chiorzolamide, a sulfonamide. Solutions of
purified enzyme (at 2.4 x 10-^ M) were irradiated at 280 nm and the
emissions were recorded at 330 nm (Perkin-Elmer MPF-44B
spectrophotometer using cells of 1.0 cm pathlength). Solutions
contained 50 mM buffer (see Figure Legends), 1.0 mil dithiothreitol to
prevent dimer formation, and no sodium sulfate. Titration with


62
chlorzo!ami de was carried out by repeated additions of a chiorzolamide
solution into enzyme solution; dilution at the end of the titration was
less than 2% of the total volume of the enzyme solution (2.5 ml). The
observed intensities of fluorescence were corrected for self-absorption
of incident light at 280 nm by chlorzolamide using the relationship in
Equation 4. The correction due to absorption by chlorzolamide of light
emitted at 330 nm was negligible. In experiments involving both
chlorzolamide and anion (CN0~), the anion was added first at the
desired concentration prior to repeated additions of chlorzolamide. In
all experiments the dissociation constant of inhibitor is much greater
than the enzyme concentration; hence, no correction was made for the
amount of chlorzolamide bound to the enzyme's active site.
Data are presented and were analyzed using this Eadie-Hofstee form
of the Langmuir equation
(11)
diss
in which AF is the difference in fluorescence intensity of protein in
chl or
the absence and presence of chlorzolamide at concentration C,
is the apparent dissociation constant for chlorzolamide and
for the anion. Values of Kc¡1-ss were determined by least-squares fits
of the slopes of plots of AF versus AF/C.
Results
The inhibition by sodium azide of the hydration of CO2 catalyzed by
cat carbonic anhydrase III at pH 6.0, 7.0 and 9.5 is shown in Figures
11, 12 and 13. These data were fit by a least-squares method (22,23)
to Equation 5.


Figure 11. Double reciprocal plot showing the inhibition by NaM3 of
the hydration of CO2 catalyzed by cat carbonic anhydrase
III (3.1 x 10" M). Temperature was 25C and 20 mM buffer,
Mes, was used and Na2S04 was present at 33 mM. Inhibition
at pH 6.0 caused by NaN3 at (a) 3.0 x 10-5 M, (), 6.3 x
10 M and (o) in the absence of NaN3. Lines were
determined by weighted least-squares analysis (see text)
which resulted in the following constants (mean and
standard deviation): k£at = (2*4 0.3) x 10^ s~l,
CO2 int r
Kra = 5.1 0.6 mM, K¡ = (2.4 0.3) x 10"5 M.


6.0
O
"To zcT
[co2](m'I) x i02
3.0


Figure 12. Double reciprocal plot showing the inhibition by NaM3 of
the hydration of CO2 catalyzed by cat carbonic anhydrase
III (3.0 x 10"6 M) at 25C. Ionic strength was the sum of
that contributed by 20 mM Mops and 33 mM Na2S04. pH was
7.0 and NaN3 was present at () 1.0 x 10"^ M, (a) 4.0 x
10-5 and (o) in the absence of MaN3. Least-squares
analysis gaye: kca£ = (1.4 0.1) x 10J sec-1, Km = 7.2
0.9 mM, Ktnt = (2.0 0.2) x 10"5, Krlope = (6.8 2.2) x
10-5 M.


^-(M-'s)xlCr


Figure 13. Double reciprocal plot showing the inhibition by NaNg of
the hydration of CO2 catalyzed by cat carbonic anhydrase
III (3.8 x 10_6 M) at 25C. Ionic strength was the sum of
that contributed by 20 mM buffer, Ted and 33 mM NagSO/j. pH
was 9.5 and MaN3 was present at (a) 1.0 x 10~3 M, and (o)
in the absence of NaNg. Least-squares analysis gave:
CO? , CO? int
kcat = (1-6 0.2) x 104 sec"1, Km = 31 3 mM, KT =
(2.5 0.3) x 10-4 m.


3.0
T
2.0
s) x I0"2
1
T
cr>
oo


69
The values of p! Figure 14 and and for potassium iodide in Table 5. At pH values 6 and
9.5, the inhibition patterns were close to uncompetitive (Figures 11
and 13) with characteristic parallel lines in a double reciprocal plot.
In the pH range 7.7 to 9.0 however, the patterns of inhibition were
mixed/noncompetitive (Figure 12). The pattern of inhibition by KI at
pH 6.0 was also close to uncompetitive. Above pH 8.0, it was
mixed/uncompetitive. The values of Kj for these anionic inhibitors
were independent of pH from 6.0 to 8.0, although Kj^0Pe varies greatly.
Both Kj1ope and l Table 5). From the analysis of data in Figure 14 using equation 12 by
a least-squares method (22,23), the pKa of the group which affects the
inhibition by Na^ of the hydration of CO2 catalyzed by cat isozyme III
was obtained as 8.4 0.3
,/intri nsic
KI
[H+]
(12)
[H+] + K
d
where | assumes that the inhibitor can bind to enzyme when a basic amino acid
residue of isozyme III is protonated. The inhibition by Na^ of CO2
hydration catalyzed by cat isozyme III gave a linear Dixon plot at pH
7.1 for concentrations up to 1.0 x 10-^ M in NaNg; however, the Dixon
plot was nonlinear at higher concentrations of NaNj (data not shown).
c h 10 r
In Figure 15, the values of the equilibrium dissociation
constant of chi orzo!amide and cat carbonic anhydrase III are given as a


Figure 14. The pH dependence of pK¡nt (open symbols) and pK^Pe
(filled symbols) for the inhibition by NaN3 of the
hydration of CO2 catalyzed by carbonic anhydrase III from
cat muscle at 25C. Solutions contained 33 mM Na2S04 and
20 mM of one of the following buffers: ( (o) Mes, (0) Mops, (a) Hepes, (v) 1,2-dimethyl imidazole,
(a) Ted. The values of pKf^^e at pH 6.0 and 9.5 have very
large experimental uncertainties and are shown here to
emphasize that they are much smaller in magnitude than
pKint.


5.0
T
pH


72
TABLE 5
THE INHIBITION BY IODIDE OF THE HYDRATION OF
C02 CATALYZED BY CAT CARBONIC AMHYDRASE III
pH
i nt
pKj
si ope
pKj
6.2
3.3
1.6
6.7
3.0
2.1
7.3
3.0
2.5
7.7
2.9
2.1
9.5
1.9
1.2
Experiments were performed at 25C in solutions
containing 0.033 M Na2S04. The buffers were 20 mM 3,5-
lutidine (pH 6.2), 20 mM Mops (pH 7.3), 15 mM Pipes (pH 6.7),
15 mM Hepes (pH 7.7) and 20 mM Ted (pH 9.5). Values of K¡
are given in the units of molarity.


Figure 15. The pH dependence of pKdjSsr for the interaction between
chiorzolamide and cat carbonic anhydrase III (2.4 x lO"? M)
determined by fluorescence quenching. Temperature was 25C
and solutions contained, in addition to enzyme, 1.0 mM
dithiothreito! and 50 mM of one of these buffers: (o) Mes;
(v) 1-methyl imidazole; (o) Mops; (o) 1,2-dimethyl imidazole;
(a) Hepes.


-p*


75
function of pH in the pH interval from 6.0 to 8.5. As can be seen, the
values of were independent of pH in the pH range 6.0 to 7.5 but
increased above pH 7.5. The p'
of chiorzolamide to cat isozyme III was obtained as 7.5 0.1 by using
Equation 12. The inhibition by the sulfonamide ethoxzolamide of the
hydration of CO2 catalyzed by cat isozyme III was measured at pH 7.7
and observed to be indistinguishable from noncompetitive (data not
shown). The inhibition pattern was also noncompetitive when
"i n t
chiorzolamide was used as the inhibitor at pH 7.7 with value of Kj
1.5 x 10 M. The binding of cyanate at pH 6.0 was inhibited in a
competitive manner by binding of chiorzolamide (Figure 16). When
experiments were performed using human isozyme II, a competitive
pattern of interaction v/as observed at pH 6.2 between the fluorescent
sulfonamide dansylsulfonamide and azide with values of Kcj1*ss 2.0 x
10"^ M and 5.0 x 10"^ M for dansylsulfonamide and H3 respectively (data
not shown). The binding of dansylsulfonamide to cat isozyme III was
too weak to be useful in these studies.
Discussion
The uncompetitive pattern of inhibition by N3 and I" of the
hydration of C02 catalyzed by cat carbonic anhydrase III at pH 6.0 can
be compared with the observation of Pocker and Oeits of uncompetitive
inhibition at pH > 8 by anions of the hydration of C02 catalyzed by
isozyme II (52). In the previous Chapters, I have argued that isozyme
III from cat similar to isozyme II has a rate-limiting proton transfer
occurring outside the interconversion of C02 and HCO3. The
uncompetitive mode of inhibition by anions of carbonic anhdyrase II was


Figure 16. Eadie-Hofstee plot of the change in corrected fluorescence
intensity at 330 nM (see text) of cat carbonic anhydrase
III was a function of concentration of chiorzolamide, a
quencher and specific inhibitor. Concentration of enzyme
was 2.4 x 10-7 pH was 6.0 at 25C. In addition to
enzyme, solutions contained 1.0 mM dithiothreitol and FO mM
Mops and KNCO at one of these concentrations: (o) 0 M; (v)
2 x 106 M; (o) 4 x 10~6 M; (a) 8 x 10"6 M; () 1.6 x
105 M.


% AForr / [chlorzolamide] (M"1 ) x ICT7


78
explained by using the enzymic model in Scheme 1 with its rate-limiting
proton transfer (7,32). I suggest that the uncompetitive type of
inhibition observed in this study could also be the result of such
rate-limiting proton transfer in the kinetic mechanism of cat isozyme
III. Since an uncompetitive pattern of inhibition for isozyme II was
observed only at pH values greater than the p
controlling group (pKa 7 for zinc-bound H2O), at pH 6.0 carbonic
anhydrase III must be in its high pH form to give the same pattern of
inhibition (Figure 11). In other words, zinc-bound H2O in cat isozyme
III could have a p
inhibition toward mixed/noncompetitive between pH 7.7 and 9 is not
understood. One possibility is that ionization of a group such as
Lys 64 is responsible.
It is interesting to note that although isozyme III and II share a
similar type of inhibition in their high pH arms, the magnitude of
inhibition constants for anions is similar for isozyme I and III (see
Table 2 in ref. 14). It is found for isozymes I and II, as pH
increases the values of Kj for anions increase, perhaps because of
the competition between OH" and anions at the active site (55). When
pH is lowered, anions can bind more strongly to the zinc H2O form of
the active site. It is therefore curious that low values of Kj are
observed for the inhibition by anions of the hydration of CO2 catalyzed
by isozyme III at pH values where the zinc-OH form the active site is
suggested to exist (Figure 14). This result could be due to the
stabilization of the negative charge on zinc-bound hydroxide by the
positive charges of the basic amino acid groups such as lys 64 and


79
arg 91. Therefore, at pH values lower than the pKa of these residues
(pH < 3), anions could coordinate to a fifth ligand site on zinc
without displacing 0H and give rise to pH independent values of Kjn^.
BH+ BH+ B
Zn OH + I" Zn OH Zn OH
I"
(13)
However, at pH > 8, the ionizations of these positive charges will
leave the negative charge on hydroxide ion unstabilized, therefore the
values of K¡ will increase (Figure 14). A value of pKa = 8.4 + 0.3
obtained from the analysis of Figure 14 is consistent with the pKa of a
group such as lysine. Further, this group may be the same one which
CO?
causes the increase in kca^ above pH 8 (Figure 6, upper panel).
The Dixon plot of azide inhibition is nonlinear above 1.0 x 10^ M
concentration of Nal^, meaning that the inhibitor bound enzyme still
has a residual activity. Tibell et jil_. (55) for isozyme II also
observed that anions give rise to nonlinear Dixon plots at
concentrations much higher than their corresponding Kj values. They
explained this result by proposing a fifth coordination site for anions
on isozyme II. Such a hypothesis was also suggested above to explain
i nt
the pH profile of K c for azide (Figure 14). Therefore, the nonlinear
Dixon plot could be further evidence for the presence of a five
coordinated anion-isozyme III complex.
The values of are also independent of pH in the pH region
6.0 to 7.5 but increases above pH 7.5 (Figure 15). This result could
also be explained by the same hypothesis given above for Figure 14.
The value of pKa 7.5 1 obtained from Figure 15 is quite lower than


so
the value of pKa 8.4 + 3 obtained from Figure 14. This difference
could be due to ionizations of the inhibitor itself (pKa values of
chiorzolamide are 6.5 and 8.8 [56]), in addition to ionizations on the
enzyme which could alter the interaction of chiorzolamide with the
active site zinc and hydrophobic region.
The noncompetitive pattern of inhibition by ethoxzolamide and
chlorzolamide of the hydration of C02 catalyzed by cat isozyme III is
similar to that observed using isozymes I and II. This noncompetitive
pattern of inhibition may be the result of tight binding by
sulfonamides Also similar to that found for isozyme I and II
(57,58), anions and sulfonamides compete for the same binding site,
metal ion in the active site of cat isozyme III.
In summary, the uncompetitive pattern of inhibition by N3 and I" of
the hydration of C02 catalyzed by cat isozyme II is consistent with the
hypothesis given in Chapter IV in that this isozyme was suggested to
have an activity controlling group with a p!
rate-limiting step located outside the interconversion of C02 and HCO3.
The pH profiles of Kjnt and K^¡5r suggested that cat isozyme III could
have a fifth coordination site for anions and sulfonamides. They also
indicated the presence of a basic amino acid group with a value of pKa
higher than 8.0.


CHAPTER SIX
CONCLUSIONS
I have shown further evidence to support the hypothesis by Tu et
al. (11) that Cu2+, through interacting possibly with the histidine 64
residue of human carbonic anhydrase II, inhibits only the
intramolecular proton transfer in the hydration and dehydration
reactions catalyzed by carbonic anhydrase II. This conclusion further
implies that the histidine 64 residue of isozyme II is also the group
which shuttles protons between the water ligand on zinc and the buffers
in solution during the catalysis of the hydration reaction.
I have shown that, while carbonic anhydrase III from cat has a
rate-limiting proton transfer located outside the interconversion of
CO2 and HCO3, it does not have any buffer effect in its catalysis of
the hydration of CO2 as found for isozyme II (1). The pH-rate profiles
of the hydration, dehydration and esterase activity of cat isozyme III
suggested that the pKa of water, which is normally 14, can be lower
than 6.0 when it is bound to the active site of cat isozyme III. I
have also discovered that a basic amino acid residue near the active
site with a pKa near 9.0 affects catalysis and also the binding of
anions and sulfonamides. Although there is no experimental evidence, I
suggest that lys 64 and arg 91, residues which are found in the active
site of bovine isozyme III which also seem to exist in cat isozyme III,
can be responsible for these effects. These basic residues, by
81


82
stabilizing the negative charges at the active site, can also decrease
the p!
Perhaps one of the reasons for carbonic anhydrase III being the
slowest isozyme of carbonic anhydrase relates to the fact that it has
these basic amino acid residues at its active site which cannot accept
a proton from the zinc-bound HpO as fast as histidine 64 can in isozyme
II, at least at pH < 8 during its catalysis. These positive charges at
the active site can also be the reason why isozyme III can bind tighter
to anions and not to sulfonamides.
Finally, I hope that this study, which establishes the similarities
and differences between the kinetic mechanisms of carbonic anhydrase II
and III, will be useful for the future studies of investigators in this
field.


APPENDIX A
History, Distribution and General Properties of
Carbonic Anhydrase from Red Cells
In the early 1930's it was already known that the release of CO2
from hemolyzed blood occurs more rapidly than expected from the uncat
alyzed rate of conversion of HCO3 to CO2. Calculations based
on the uncatalyzed rates indicated that one passage time of blood through
lungs would only permit the release of 17% of CO2 known to be excreted
(59). In 1933 Meldrum and Roughton discovered carbonic anhydrase in red
blood cells as a discrete protein different than hemoglobin (60). The
presence of carbonic anhydrase activity in pancreas and muscle was also
reported by the same investigators (60). Anions, cyanide, sulfide and
metal ions Cu^+ and Hg2+ were shown to be inhibitors of this
enzyme (60). The role of carbonic anhydrase in respiration was elegantly
explained by Roughton in his 1935 review article (59). In 1940 Keilin
and Mann, after purifying carbonic anhydrase some 200 times, showed the
presence of zinc, hence made carbonic anhydrase the first zinc enzyme to
be recognized (61). Mann and Keilin also discovered that sulfanilamide
is a specific inhibitor of carbonic anhydrase with a K¡ 10"^ M
(62). Mann and Keilin went on further and showed that all unsubstituted
aromatic sulfonamides R-SO2-NH2 are active (62). Roblin and Clapp
(63) and Miller et al. (64) synthesized dozens of unsubstituted hetero
cyclic sulfonamide compounds which have higher inhibitory activities than
sulfanilamide. From these compounds, acetazolamide, ethoxzolamide, meth-
azolamide and benzolamide became the most valuable tools in research in
83


84
determining the role of carbonic anhydrase in transport of metabol-
ically formed CO2 and production and secretion of H+ and HCO3
in blood, kidney, pancreas, stomach, liver, CSF, eye, lung and many
other organs (65). Maren discovered that for a sulfonamide inhibitor
to exert any pharmacological effect, it must be present at concentra
tions high enough to inhibit 99.9% of enzyme in kidney and many
tissues, because the concentration of carbonic anhydrase in tissues is
in excess and CO2 hydration is never rate-limiting (65,66).
In 1960 Nyman (67) and Lindskog (68) purified for the first time
the isozymes of carbonic anhydrase from erythrocytes. Human erythro
cytes contain the B (or I) and C (or II) forms of carbonic anhydrase.
Isozyme C is one of the fastest enzymes in nature and has about five-
C0?
fold larger k £ for hydration of CO2 (Table 1) than that for
isozyme B. Both isozymes have about 260 amino acid residues in a
single polypeptide chain (isozyme C has one less) and M.W. of 30,000
(69). There is a 59% homology between the amino acid sequences of
isozymes B and C (70). Two forms of carbonic anhydrase were isolated
from bovine erythrocytes (68) and both have the same kinetic and
molecular properties, similar to that of isozyme C from human red cells
(68). Although letters B and C and Roman numerals I and II were
interchangeably used in the past to name the red cell isozymes, after
the discovery of the third isozyme in muscle now only Roman numeral
notations are used in the genetic literature (71).
Distribution of isozymes I and II in mammalian tissues has been
reviewed by Maren (65) and recently by Tashian et al. (72). It appears
that isozyme II occurs more widespread than isozyme I especially in
secretory tissues. Carbonic anhydrase I is the most abundant protein


8b
in red blood cells of man, also found in primates and rodents but mis
sing in the red cells of ruminants and felines (65). Maren et al. (73)
have pointed out the very powerful inhibitory action of Cl" ions on
carbonic anhydrase I, at the chloride concentrations present in the red
cells. Therefore the function of isozyme I in red cells is not clear
yet. Besides in vertebrates, carbonic anhydrase is also found in
invertebrates, higher plants, algae and bacteria (75). Carbonic
anhydrase in cells can appear either as cytosolic protein or bound to
membrane (65,74).
Although physiological substrates for carbonic anhydrase isozymes
are CO2 and HCO3, they can also catalyze the hydrolysis of
esters and hydration of aldehydes (75).
Studies on the kinetic and spectroscopic properties of mammalian
carbonic anhydrase I and II have been very intense in the laboratories
of Lindskog, Pocker, Coleman, Silverman, Koenig and Bertini. Here I
shall not describe any of their findings, but lead readers to excellent
recent reviews by Pocker and Sarkanen (75), Lindskog (76) and Silverman
and Vincent (31).
History, Distribution and Physiological Function
of Carbonic Anhydrase III
The presence of CO2 hydration activity in the watery extracts of
mammalian muscles which were perfused well with saline solution to
remove the blood was reported as early as in 1933 by Mel drum and
Roughton (60). Despite this observation, Roughton stated that "in such
a location carbonic anhydrase would be an enemy to the organism, rather
than a friend: in its absence the CO2 is able to diffuse away
rapidly without loitering appreciably by the wayside in the form of


36
HCO3 ions" (59). This statement perhaps delayed for several
decades the discovery of carbonic anhydrase in muscle. Another factor
which also contributed to such a delay was the expectation that the
hydration of CO2 catalyzed by carbonic anhydrase should be inhibit-
able by 10"? to 10" M concentrations of sulfonamide inhibitors
such as acetazolamide. We now know that carbonic anhydrase III is
rather resistant to inhibition by several sulfonamide inhibitors. Lack
of inhibition by 10"? to 10 M of acetazolamide was inter
preted as the absence of carbonic anhydrase in muscle. Maren et al. in
1966 (77) reported that the supernatant solutions of male rat liver
extracts have carbonic anhydrase activity which is about 10 to 3000
times less sensitive to inhibition by sulfonamides than seen in the red
cell or kidney preparations. In 1974 purification of that sulfonamide-
resistant carbonic anhydrase from the male rat liver was reported by
King et al. (78). It was found that this enzyme is not only quite
resistant to inhibition by sulfonamide inhibitors 'out also has a turn
over number for hydration of CO2 which is about 1% of that red cell
isozyme II (78). At that time it was clear that this liver carbonic
anhydrase is different than red cell isozymes I and II. Holmes in 1976
reported the discovery of carbonic anhydrase III from the skeletal
muscles of sheep, chicken, rabbit and mice (79), and subsequently puri
fied this enzyme from the skeletal muscle of sheep and chicken (12).
In the same year in Noltmann's group identification of the so called
"basic muscle protein" as carbonic anhydrase III was established (13).
This basic muscle protein v/as first recognized in 1972 during the puri
fication procedures of phosphoglucose isomerase from rabbit muscle and


87
at that time its function was not understood (80). So far, purifica
tion of this third isozyme of carbonic anhydrase has been reported in
sheep, chicken, horse, rabbit, bovine, cat, pig, human/gorilla skeletal
muscles (12-14,81-84,39). Isozyme III has also a zinc ion essential
for its activity and a molecular weight of about 30,000 similar to
other isozymes (12,13). It originates from a genetic locus distinct
from those responsible for the synthesis of red cell isozymes I and II
(39). The amino acid composition of isozyme III showed some similar
ities to red cell isozymes (81), moreover Tashian's group has shown
that for bovine isozyme III there is considerable sequence homology
with the isozymes I and II, though there appear to be two arginyl resi
dues and one lysyl residue substituting for three of the active site
residues in isozymes I and II (39). Partial sequences of muscle iso
zyme from bovine, human, chicken and rat revealed almost identical res
idues at positions from 79 to 92 (85). Depending on the species its
isolated from, isozyme III has 2 to 6 cysteine residues (81) which have
been shown to cause dimerization through disulfide bonding formation
(12,13). The concentration of isozyme III in skeletal muscle in the
cat is high, about 100 ym constituting 1 to 2% of the total soluble
cytosolic proteins (14). Histochemical methods confirm its presence in
the cytoplasm of muscle cells (86,87).
An interesting study by Carter et al. showed that carbonic anhy
drase enzymes isolated from male rat liver and skeletal muscle have
identical amino acid sequence, immunodiffusion and electrophoretic
patterns (85). Moreover, the inhibitory activities of both anions and
sulfonamides against purified male rate liver and cat muscle carbonic
anhydrase III were shown to be similar by Sanyal et al. (14).


88
The physiological function of muscle carbonic anhydrase III is un
known. Holmes suggested that this enzyme may function in CO2/HCO3
exchange and transport (12). However, the results of Sanyal et al.
which showed ^ values for cat isozyme III as high as 30 mM
do not support such an hypothesis (14). Its possible function as a phos
phatase is also unlikely, because of the extremely low values of Km for
this reaction (33). Its role in intracellular buffering during acidosis
and alkalosis was also considered (14), but is not supported due to its
absence in tissues such as cardiac muscle (88) which has considerable
buffering capacity (89,90), and its presence in tissues such as skeletal
muscle (12,13) where carbonic anhydrase III occurs at high concentra
tions, but there is very little buffering capacity (91).
Despite Roughton's statement which I have quoted at the beginning of
this section, Sanyal et al. (14) suggested that isozyme III in muscle
might facilitate diffusion of metabolically formed CO2 out of muscle.
There is now a good body of evidence to support such an hypothesis. Gros
et al. in their study showed that carbonic anhydrase is necessary for
facilitated diffusion of CO2 to occur in phosphate buffer solutions at
low pC2 gradients (92). Moreover Kawashiro and Scheid showed that
facilitated diffusion of CO2 indeed occurs in muscle preparations (93).
But unfortunately they have not shown the effect of carbonic anhydrase
inhibition on this facilitated diffusion (93). Studies in Maren's lab
showed that the role of carbonic anhydrase on the lens (94) and rectal
gland (95) appears to be for the carriage of metabolically formed CO2.
Inhibitors of carbonic anhydrase cause accumulation of CO2 in these
tissues indicating that the transport of CO2 is diminished in the


89
absence of carbonic anhydrase. These studies indicate that a possible
role of carbonic anhydrase III in muscle for the facilitated diffusion of
CO2 is quite likely.
Enns and Hill (96) and Geers and Gros (97) in a similar kind of
study showed that the washout of from the bloodless hindlimb of
cats and rabbits considerably increases when carbonic anhydrase inhib
itors are present in the perfusate. This effect was due to decreased
entry of labelled CO2 and HCO3 into tissues after carbonic
anhydrase inhibition. Geers and Gros showed that the onset of action by
sulfonamides, methazolamide and benzolamide, with differing lipid solu
bility was less than 1 minute, also the IC50 of these inhibitors were
inconsistent with their known values for pure isozyme III (97)
(IC5o those effects seen with sulfonamides were not due to inhibition of the
intracel1ularly located carbonic anhydrase III, but rather due to inhibi
tion of another type of enzyme bound from the outside to muscle cell mem
brane or endothelium (97).
So far these studies bring little insight to what carbonic anhydrase
III might be doing in skeletal muscle. Further experiments are necessary
in which selective carbonic anhydrase II and III inhibitors should be
used in resting and exercising muscle to understand the role of isozyme
III.


APPENDIX B
The computer simulation experiments conducted here are an extension
of the previous work by Rowlett and Silverman (32) which uses the
enzymic model shown in Scheme 1. The details of these computer
simulation experiments are given by Rowlett and Silverman. Here, a
brief description will be presented.
The change in the concentrations of each enzyme form with time can
be described by a set of differential equations. Equation 14
demonstrates one of those differential equations for the E form of the
enzyme in Scheme 1.
= k2[EX] + k7[EH] [B] kx[S] + k8[BH][E] (14)
Then this set of differential equations is solved by the Euler method
of numerical integration (98) to obtain the steady-state concentrations
of each enzymic species.
This method involves taking very small increments of time dt to
calculate the rate of change of each enzyme species at time t and then
updating the concentration of each enzyme form after each iteration.
When all of the enzyme forms have reached their steady-state
concentrations, the initial rate of hydration is calculated from the
rate of appearance of HCO3 as given by Equation (15).
JF^- = k3CEX + HEX] ^HCO^iCEH] + [HEH]) (15)
90


91
These simulation experiments by Rowlett and Silverman (32)
explained almost all the kinetic features of carbonic anhydrase II,
some of which are mentioned in Chapter One.
In this study, I have modified the computer programs by Rowlett and
Silverman on the basis of the new model given in Scheme 2. This model
O i
is the same as the one given in Scheme 1, except it incorporates Cu
as a specific inhibitor of carbonic anhydrase II (bottom cage of
pi
Scheme 2) and assumes that Cu inhibits only the intramolecualr proton
transfer event through binding to the histidine 64 residue of this
isozyme. The values of the on and off rate constants of Cu were
calculated as 1.0 x 10^ M"-*- sec--*- (diffusion limited value) and 5.0 x
3 -1 p. .2+ _7
10 sec on the basis of K^ss = 5.0 x 10 M.
The results of these simulation experiments which are conducted
under different reaction conditions are summarized below.
1. At fixed buffer concentration (15 mM) when the concentration of
CO2 was varied from 6 to 30 mM, the resulting patterns of inhibition by
? +
Cu of the hydration of CO2 catalyzed by isozyme II were
noncompetitive for values of pH from 6.0 to 8.0 (Figure 17).
2. The values of Kjn1: or Kj^Pe which decreased as pH increased
pi
gave a pKg value 7.0 for the group to which Cu binds (Figure 18).
3. The corresponding pattern of inhibition was uncompetitive when
CO2 concentration was fixed (8.5 mM) and buffer concentrations were
varied from 1 to 10 mM at pH 7.0 (Figure 19).


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