(Received for publication, November 18, 1996, and in revised form, March 18, 1997)
From the § Department of Pharmacology and Therapeutics
and the Department of Biochemistry and Molecular
Biology, University of Florida College of Medicine,
Gainesville, Florida 32610-0267
A cDNA encoding the murine carbonic anhydrase
IV (mCA IV) gene, modified to resemble a form of mature human carbonic
anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X. L., and
Sly, W. S. (1995) Arch. Biochem. Biophys. 320, 315-322),
was expressed in Escherichia coli. Inactive inclusion
bodies were collected and refolded, and active enzyme was purified; the
resulting mCA IV was used to characterize the catalysis of
CO2 hydration using stopped flow spectrophotometry and
18O exchange between CO2 and water. Unlike
previously studied isozymes in this class of carbonic anhydrase, the pH
profile for kcat for hydration of
CO2 catalyzed by mCA IV could not be described by a single
ionization, suggesting multiple proton transfer pathways between the
zinc-bound water molecule and solution. A role for His64 in
transferring protons between the zinc-bound water and solution was
confirmed by the 100-fold lower activity of the mutant of mCA IV
containing the replacement His64 Ala. The remaining
activity in this mutant at pH levels near 9 suggested a second proton
shuttle mechanism. The maximal turnover number
kcat for hydration of CO2 catalyzed
by mCA IV was 1.1 × 106 s
1 at pH > 9. A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.
The mammalian carbonic anhydrases
(CAs1) constitute a gene family of at least
seven distinct isozymes (1) that catalyze the hydration of
CO2 to form bicarbonate and a proton: CO2 + H2O HCO3
+ H+. Although
these isozymes are characterized by a high degree of amino acid
identity (28-59%) (2), they are quite diverse in their cellular
distribution, catalytic activity, and physiological function (reviewed
in Ref. 3). Among these isozymes, CA IV is the only known
membrane-associated form. It was first identified and purified from
bovine lung (4), although the presence of a membrane-bound carbonic
anhydrase activity had been observed earlier (reviewed in Refs. 5 and
6). Subsequent purifications from human kidney (7, 8), human lung (8),
and lung microsomal membranes from a variety of mammals (9) identified
CA IV as a 35-52-kDa protein anchored to the membrane by a glycosyl
phosphatidylinositol linkage to its C terminus. The
distribution of membrane-associated carbonic anhydrases is widespread;
they have been found in many secretory tissues, where they play a
prominent role in, for example, the formation of ocular fluid,
cerebrospinal fluid, and other secretions (reviewed in Ref. 10).
Moreover, CA IV is the luminal CA in the proximal tubule of the kidney
and is estimated to mediate 85% of renal bicarbonate reabsorption
(10). Membrane-associated carbonic anhydrases have been found in many
other tissues including the capillaries of skeletal and cardiac muscle,
the colon, and the reproductive tract.
The crystal structure of human CA IV shows considerable backbone similarity to that of CA II, especially in the region of the active site (11). Two disulfide bridges appear in the structure of human CA IV (11) that are not present in CA II; these are likely responsible for the enhanced stability of CA IV against heat and SDS. Whitney and Briggle (4) reported that CA IV, unlike the other isozymes, was stable for several hours in 1 to 5% SDS, an observation that has been used to facilitate its purification.
Measurements near physiological pH have identified human (7) and bovine
CA IV (12) as a fast isozyme, with a catalytic turnover
kcat of approximately 2 × 105
s1 at 0 to 1 °C, close in magnitude to that of CA II,
the most efficient of the carbonic anhydrase isozymes. Early studies
showed that the membrane-bound carbonic anhydrase from the brush border
of the dog kidney had catalytic constants nearly identical to those of
carbonic anhydrase II, suggesting that adherence to the membrane did
not significantly diminish the activity to below that of CA II (13).
Sulfonamides inhibit CA IV, but the average inhibition constant is
17-fold less than for CA II (12).
cDNA clones for human (14), rat (15), and mouse (16) CA IV have been isolated. Until recently, obtaining sufficient amounts of CA IV for kinetic and other studies was limited to the enzyme produced from tissue isolation or overexpression in a mammalian cell line (17). Refolding of CA IV inclusion bodies obtained from expression in Escherichia coli2 have produced sufficient quantities of active, SDS-resistant, murine carbonic anhydrase IV (mCA IV) to characterize its kinetic constants in the hydration of CO2 over a range of 4-5 pH units, allowing an examination of the mechanism of catalysis including the pKa of the zinc-bound water molecule. In this study we present the full pH profiles for catalysis of the hydration of CO2 by mCA IV using both initial velocities measured by stopped flow and 18O exchange between CO2 and water measured by mass spectrometry. The data show a very efficient enzyme but with features that are unique among the studied isozymes of the human and animal carbonic anhydrases; the maximal turnover appears to be dependent on at least two ionizations, suggesting that more than one proton shuttle group is functioning in the catalytic pathway. One of these is a shuttle group with a pKa near 7, which is identified as His64 by site-specific mutagenesis. These data are discussed in the context of a number of recent cases suggesting the role of multiple proton transfers in catalysis and proton-translocating proteins.
A murine Balb/c
lung carbonic anhydrase IV cDNA was isolated,2 and a
portion of the coding sequence (see legend to Fig. 1) was expressed in
E. coli strain BL21(DE3)pLysS using the pET31 T7 expression
vector described by Tanhauser et al. (18). The mutant H64A
mCA IV was constructed using a mutating oligonucleotide (18, 19) and
verified by DNA sequencing. The bacterial cells were lysed, and mCA
IV-enriched inclusion bodies were isolated, denatured in guanidine
hydrochloride, and refolded into an active form.2 Further
purification was carried out using a combination of gel filtration
(Ultrogel AcA 44, LKB) and ion exchange (DEAE-Sephacel, Sigma)
chromatography (20). The purity of the isolated enzyme was estimated at
greater than 95% by electrophoresis on 10% polyacrylamide gels. The
concentration of active carbonic anhydrase was determined by titration
with the carbonic anhydrase inhibitor ethoxzolamide (Ki = 16 nM) while observing the
catalyzed 18O exchange between CO2 and water;
more than 96% of the enzyme was active, indicating that nearly all of
the purified mCA IV had been refolded into the active conformation.
18O Exchange
The catalyzed and uncatalyzed
rates of 18O exchange from species of CO2 into
water and the rates of exchange of 18O between
12C-containing and 13C-containing species of
CO2 were measured at chemical equilibrium using a mass
spectrometer. Equations 1 and 2 demonstrate the catalytic pathway for
the exchange of 18O from bicarbonate to water. In Equation 2, B is a buffer in solution and/or an amino acid side
chain in the enzyme.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
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![]() |
(Eq. 3) |
The second rate determined by this method is RH2O, the rate of release from the enzyme of water labeled with 18O (Equation 2). A proton donated from a donor group BH converts the zinc-bound hydroxide to zinc-bound water, which readily exchanges with unlabeled water. The 18O label is effectively lost by dilution into the solvent water. The value of RH2O can be interpreted in terms of the rate constant from a predominant proton donor group to the zinc-bound hydroxide according to Equation 4 (23), in which kB is the rate constant for proton transfer to the zinc-bound hydroxide, KB is the ionization constant for the donor group, and KE is the ionization constant of the zinc-bound water molecule.
![]() |
(Eq. 4) |
A stopped flow spectrophotometer
(Applied Photophysics Model SF.17MV) was used to measure initial
velocities of the hydration of CO2. Since this catalysis
produces protons as well as HCO3, we measured the
initial rate of hydration by recording the absorbance change of a pH
indicator (24). Saturated CO2 solutions were made by
bubbling CO2 into water at 25 °C. Syringes with
gas-tight seals were used to make CO2 dilutions from 17 to
0.24 mM. The pKa of the buffer indicator
pairs, and the observed wavelengths, were as follows: Mes
(pKa 6.1) with chlorophenol red (pKa 6.3, 574 nm); Mops (pKa 7.2)
with p-nitrophenol (pKa 7.1, 400 nm);
Hepes (pKa 7.5) with phenol red
(pKa 7.5, 557 nm); Taps (pKa 8.4)
with m-cresol purple (pKa 8.3, 578 nm);
and Ches (pKa 9.3) with thymol blue
(pKa 8.9, 590 nm). The buffer concentration was 25 mM, unless indicated otherwise, and the total ionic
strength for each buffer-indicator pair system was maintained at 0.1 M by the addition of the appropriate amount of
Na2SO4. Solutions contained 4 µM
EDTA. The mean of four to eight reaction traces of the first 5 to 10%
of the reaction was used to determine initial rates. The uncatalyzed
rates were subtracted, and the rate constants kcat and
kcat/Km were determined by
nonlinear least squares methods (Enzfitter, Elsevier-Biosoft). The
values of Km for hydration of CO2 were
below 10 mM at pH < 8.5 and reached a maximal value
near 20 mM at pH approaching 10; hence, we were able to
achieve concentrations of CO2 approaching saturation over most of the pH range studied. The S.E. values in
kcat and
kcat/Km were ±8% at
most.
The catalysis by mCA IV of the
hydrolysis of 4-nitrophenyl acetate was measured by following the
increase in absorbance at 348 nm, corresponding to the isosbestic point
of nitrophenol and the nitrophenolate ion using the molar absorptivity
5.0 × 103
M1·cm
1 (25). Because the
catalysis of this hydrolysis by mCA IV was slow, we determined the
overall rate and subtracted from it the rate in the presence of 20 µM ethoxzolamide to inhibit mCA IV. The difference was
the component of the overall hydrolysis due to the hydrolysis at the
active site. Initial velocities were determined under the conditions
given in the legend to Fig. 4. The value of Km for
catalysis was too large to measure because it exceeded the solubility
of substrate. As a result, we were limited to observing catalytic rates
that were first order in the substrate from which we obtained
kcat/Km.
A murine CA IV cDNA coding sequence closely corresponding in structure to human CA II was inserted into a pET31 vector and protein expressed in E. coli BL21(DE3)pLysS (18). An alignment of the full-length murine CA IV protein sequence with that of human CA II is shown in Fig. 1. The two arrows in Fig. 1 show the initial and terminal amino acids of the expressed mCA IV used in this study. Mature human and rat CA IV isolated from lung have been shown to be N terminally truncated by 18 and 17 amino acids, respectively, to remove the putative plasma membrane targeting sequence (9). The murine CA IV coding sequence was truncated to mimic that of the endogenous rat protein, removing the first 17 amino acids and converting the next residue into a start methionine. The aspartate in the mouse sequence that immediately follows this methionine corresponds to the second residue in the mature rat protein (9, 15). Human CA IV is C terminally cleaved during maturation immediately after serine 266 and attached to a glycosylphosphatidylinositol anchor by this residue. This membrane anchor can be enzymatically removed without altering enzyme activity, and a fully active truncated form of human CA IV can be expressed without the residues C terminal to serine 266 (corresponding to position 258 in Fig. 1) and lacking attachment to a glycosylphosphatidylinositol anchor (17). The form of murine CA IV examined here is C terminally truncated two amino acids beyond the corresponding serine in the human sequence (17). One additional change occurred during construction of the murine CA IV expression clone; this converted a lysine at position 12 to a glutamate (Fig. 1). Residue 12 is a glutamate in human CA IV (14). Occurring in a loop region of human CA IV, Glu12 is a surface residue that extends into solvent and has no interactions with other residues in the crystal form of the enzyme (11).3 We anticipate that the presence of glutamate at position 12 has no effect on the catalysis by murine CA IV.
Catalytic ActivityThe rate constant
R1/[E] for the interconversion of
CO2 and HCO3 at chemical equilibrium
catalyzed by mCA IV was measured as a function of total concentration
of all species of CO2, with data at pH 6.7 given in Fig.
2. The dependence of
R1/[E] on the sum of the
concentrations [CO2] + [HCO3
] was
nearly linear with very little evidence of saturation. For example, the
very slight curvature in the data of Fig. 2 is consistent with a value
of KeffS of 270 ± 30 mM, where S represents all CO2
species, both CO2 and HCO3
. Because this
large value of KeffS greatly
exceeded the solubility of CO2 and HCO3
,
we were not able to obtain a value of
kcatex in Equation 3. However, we
were able to obtain
kcatex/KeffS
from the slope of plots such as the one in Fig. 2 or from studies with
([CO2] + [HCO3
])
KeffS.
The ratio kcat/Km for the
hydration of CO2 was determined by two methods, measurement
by mass spectrometry of the exchange of 18O between
CO2 and water and measurement by stopped flow of the initial velocity of CO2 hydration. When [S]
Km there is an equilibrium distribution of
enzyme forms also in steady state; hence, the ratio
kcatex/KeffCO2
(for S = CO2) obtained by 18O
exchange is in theory and in practice equivalent to
kcat/Km for hydration of
CO2 obtained in steady-state measurements (22). The
18O method was carried out at a total concentration of 25 mM of all CO2 species under conditions (given
in the legend to Fig. 3) for which ([CO2] + [HCO3
])
KeffS. This approach for catalysis
by mCA IV yielded a maximal value of
kcat/Km of 3.2 ± 0.1 × 107
M
1·s
1 with an apparent
pKa for the catalysis of 6.6 ± 0.1 (Fig. 3).
The presence of up to 200 mM imidazole in solution had no
effect on kcat/Km measured by
18O exchange (shown for 100 mM in Fig. 3). The
measurement of kcat/Km for
hydration of CO2 by stopped flow gave data with less
precision (data not shown); the analysis of these results yielded a
maximal value of kcat/Km of
5.0 ± 0.2 × 107
M
1·s
1 with an apparent
pKa of 7.3 ± 0.2. The solvent hydrogen isotope
effect on kcat/Km determined
for mCA IV by 18O exchange was
D(kcat/Km) = 0.83 ± 0.11, measured at pH 6.8 and 25 °C in solutions
containing no buffers. The 18O exchange studies extended to
catalysis by the mutant H64A mCA IV gave a maximal value of
kcat/Km of 6.3 ± 0.2 × 107
M
1·s
1 with an apparent
pKa of 7.3 ± 0.1 (Fig. 3).
The value of the apparent pKa for catalysis by mCA
IV was confirmed by measurement of the catalytic hydrolysis of 4-nitrophenyl acetate. The inherent efficiency of mCA IV in this catalysis was very low. To ensure that we were measuring catalysis at
the same active site as is involved in hydration of CO2, we determined that component of the overall rate of catalytic hydrolysis of 4-nitrophenyl acetate that was inhibited in the presence of 20 µM ethoxzolamide, a specific and potent
(Ki = 16 nM) inhibitor of mCA IV. The
maximal value of kcat/Km for this hydrolysis was 20 ± 1 M1·s
1 with an apparent
pKa of 6.5 ± 0.2 (Fig. 4).
The rate constant RH2O/[E] for the
proton transfer-dependent release of
18O-labeled water from the active site (Equation 2) was
measured by mass spectrometry in the absence of buffers; the values of RH2O/[E] as a function of pH are
shown in Fig. 5.
RH2O/[E] was evaluated by Equation 4 to determine a rate constant for proton transfer to the zinc-bound
hydroxide as well as values of pKa for the
predominant proton donor group and for the zinc-bound water molecule.
Equation 4 was fit by least squares analysis to the data for catalysis
by mCA IV (Fig. 5) to yield the rate constant for proton transfer
(kB = 1.4 ± 0.2 × 106
s1) with pKa = 6.9 ± 0.1 for the
proton donor group and pKa = 6.6 ± 0.1 for the
zinc-bound water molecule. This latter value is in agreement with the
estimate for the zinc-bound water molecule obtained by measurement of
kcat/Km for hydration of
CO2 and for ester hydrolysis. The solvent hydrogen isotope
effect on kB was
D(kB) = 1.9 ± 0.4. RH2O/[E] catalyzed by H64A mCA IV
was less than that for the unmodified enzyme by close to 100-fold (Fig.
5). The value of RH2O/[E] catalyzed
by H64A mCA IV was enhanced more than 10-fold and in a saturable manner
by the addition of imidazole with an apparent
Kmbuffer of 80 ± 20 mM
and a maximal value of 1.9 ± 0.2 × 105
s
1 at pH 7.2 (data not shown). In contrast, the value of
RH2O/[E] catalyzed by wild-type mCA
IV was enhanced 50% by addition of imidazole at the level of
saturation (50-100 mM imidazole) under the same
conditions.
The catalytic turnover number for the hydration of CO2
catalyzed by mCA IV and measured at steady state had a maximal value at
high pH of kcat = 1.1 ± 0.1 × 106 s1 (Fig. 6). This maximal
value of kcat is very similar to that of
kB determined from 18O exchange. The
pH dependence of kcat could not be fit to a
single ionization and shows the influence of another ionizable group or
an inhibition from other sources in the region of pH between 7 and 9 (Fig. 6). Data collected in the presence of 25 mM imidazole partially overcame this effect on kcat (Fig. 6),
suggesting that it is in part due to impeded proton transfer in the
active site. The turnover number kcat for
CO2 hydration catalyzed by H64A mCA IV was considerably
less than kcat for the unmodified mCA IV (Fig. 6).
Inhibition of mCA IV
The Ki values,
describing the inhibition of 18O exchange between
CO2 and water catalyzed by mCA IV, were determined from
data collected at 25 °C. For ethoxzolamide, Ki = 16 ± 1 nM; for cyanate, Ki = 97 ± 13 µM. Both of these inhibitors blocked
kcat/Km and
RH2O equally. However, cupric ion
Cu2+ blocked only RH2O with a value
of IC50 = 0.6 ± 0.1 µM (Fig.
7); the value R1/[E]
and hence of kcat/Km
determined by 18O exchange was not affected by cupric ion
concentrations as great as 40 µM (Fig. 7).
In this study we describe the catalytic properties of murine CA IV lacking an N-terminal signal peptide and C-terminal extension, an enzyme designed to resemble both the mature form of CA IV and cytosolic CA II, a well studied and efficient isozyme of carbonic anhydrase. An amino acid alignment of this murine CA IV with that of human CA II is shown in Fig. 1. The overall sequence identity of 37% includes residues involved in coordination to the zinc and in hydrogen bonding to the zinc-bound water molecule (Thr199, Glu106) that are conserved in all of the animal carbonic anhydrases. Moreover, the crystal structures of human CA IV and human CA II in the region of their active sites are very similar (11). Through these similarities, the general catalytic pathway of murine CA IV can be rationalized by analogies to isozyme II as described in Equations 5 and 6.
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
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However, despite these similarities between CA II and CA IV, catalysis by murine CA IV appears unique among the isozymes of carbonic anhydrase based on the evidence of Fig. 6 that its catalysis involves multiple proton transfer pathways. This evidence as well as the role of His64 in the proton shuttle is discussed below.
Role of His64The maximal turnover for
CO2 hydration catalyzed by CA II has been carefully
considered in previous work to be determined in rate almost entirely by
the intramolecular proton transfer from the zinc-bound water molecule
to His64 (27, 28). The similar values of
kcat for isozymes II and IV as well as the
presence and similar conformation of His64 in both of these
isozymes (11) strongly suggest that the same intramolecular proton
transfer contributes to the maximal velocity of CA IV. The maximal
turnover number kcat for the hydration of CO2 catalyzed by mCA IV at 25 °C was observed to be
1.1 × 106 s1 (Fig. 6), quite close in
magnitude to the kcat value of 1.4 × 106 s
1 measured under comparable conditions
for human CA II (24).
Further support for the role of His64 in CA IV comes from
18O-exchange studies. The rate constant for the release of
18O-labeled water from mCA IV,
RH2O/[E] shown in Fig. 5, reveals a
bell-shaped dependence on pH very similar to that of CA II (29). This
curve is fit well by the superposition of two ionizations (Equation 4)
describing the presence of the zinc-bound hydroxide and the presence of
a second group that donates a proton to the zinc-bound hydroxide, such
as the imidazolium side chain of His64. These
18O-exchange data confirm the pKa of 6.6 for the ionization of the zinc-bound water and also provide the
pKa of 6.9 ± 0.1 for the second ionization
(Fig. 5). Again this value is close to that determined by the titration
of the proton magnetic resonance of the C-2 proton of His64
in human CA II, which yields a value of pKa = 7.1 (30). These same 18O-exchange data of Fig. 5 give a maximal
rate constant for intramolecular proton transfer from His64
to the zinc-bound hydroxide molecule of kB = 1.4 ± 0.2 × 106 s1, consistent
with the maximal value of kcat, which is also
determined by this intramolecular proton transfer. That these values
should be nearly identical for proton transfer-dependent
processes in the hydration and dehydration directions is the result of
the values of the pKa for donor and acceptor
being nearly identical at pKa = 7.
The role of His64 as proton donor in CA IV is further
supported by the inhibition of catalysis by cupric ions and by the much reduced values of kcat and
RH2O for the mutant H64A mCA IV. The data for
the unmodified mCA IV showed inhibition of RH2O
by cupric ions (IC50 = 6 × 107
M), with no effect on the rate of interconversion of
CO2 and HCO3
(R1/[E]) up to a concentration of
40 µM (Fig. 7). These data are consistent with an
inhibition of the proton-transfer pathway with no effect on the
function of the zinc-bound hydroxide in the first stage of catalysis
that produces HCO3
. An analogous result was obtained
with CA II, in which RH2O was inhibited by
Cu2+ with an IC50 of 1.0 × 10
7 M and by Hg2+ with an
IC50 of 1.6 × 10
7 M with no
effect on R1 (31). This was interpreted as an
inhibition of the function of His64 as a proton shuttle and
was confirmed by the crystal structure of the Hg2+·CA II
complex showing a superposition of structures, about half with mercuric
ion bound to N
and about half bound to N
of the imidazole side
chain of His64 (32). The similar inhibition of CA IV and CA
II by cupric ions is strong evidence for the role of His64
in proton transfer by CA IV. Moreover, the inhibited value of RH2O/[E] near 2 × 104 s
1 for CA IV in the presence of excess
Cu2+ (Fig. 7) is close to the value of
RH2O/[E] for the mutant H64A CA IV
in which His64 is replaced by alanine (Fig. 5) and is close
to the value of RH2O/[E] for H64A
CA II (29), suggesting that this binding of Cu2+ has nearly
completely inhibited the proton transfer role of a shuttle group. The
remaining proton-transfer activity is due to other less efficient
proton-transfer residues of the protein or to water in the active
site.
These inhibition data suggesting a proton shuttle role for His64 were supported by the much lower values of kcat for hydration and RH2O/[E] catalyzed by the mutant H64A mCA IV (Figs. 5 and 6). Both of these rate constants are determined largely by proton transfer from or to the zinc-bound water or hydroxide (27, 29) in CA II. This is further confirmed by the significant enhancement of RH2O and kcat for H64A mCA IV by addition of imidazole, achieving a chemical rescue by providing the proton-transfer group from solution (Fig. 6).
Multiple Proton-transfer PathwaysAlthough the maximal values
of kcat for hydration are similar for mCA IV and
CA II, there is a significant difference: the pH dependence of CA II
can be described by a single ionization (24), that of the proton
acceptor His64, but the pH dependence of
kcat for mCA IV is more complex, indicating the
influence of at least two ionizable groups (Fig.
6).4 One of these groups is
His64 acting as a proton shuttle, as described above, and
the second is at least one other proton shuttle of
pKa near 9 that has not yet been identified but that
is similar to that observed in CA V (33). The activation of
kcat through chemical rescue caused by the
presence of 25 mM imidazole (Fig. 6) suggests that the
depression in the pH profile of kcat for mCA IV
near pH 7 is due to reduced capacity of the proton shuttle. That is,
mCA IV has the capacity to attain higher values of
kcat with an external proton acceptor, a result
that suggests that the function of the internal proton shuttle
His64 in mCA IV is partially blocked. One possible
explanation for the more complex pH profile of mCA IV may be the
presence of threonine at position 65, whereas human CA II has an
alanine at this position (Fig. 1). This Thr65 may block the
proton shuttle function of His64 in a manner similar to
that of Phe65 in CA V (33). However, in CA II Jackman
et al. (34) found no effect of the replacement
Ala65 Thr on the function of the proton-shuttle
mechanism involving His64. It is noteworthy that murine CA
IV has glutamine at residue 63, whereas human CA II has glycine at this
position; hence, in this position also murine CA IV has a bulkier side
chain that could hinder the role of His64 as a proton
shuttle. Tamai et al. (41) have reported evidence consistent
with the influence of residue 63 in the overall catalysis by CA IV; the
replacement Gly63
Gln in bovine CA IV decreases
catalytic activity measured in a colorimetric assay near neutral pH,
and the replacement Gln63
Gly in murine CA IV increases
activity. This role of residue 63 can also explain the differences in
the pH profile for kcat obtained for murine CA
IV in this work and that of Baird et al. (42) for human CA
IV. Baird et al. observed a pH profile for kcat catalyzed by human CA IV that is consistent
with a single ionization; at physiological pH this value of
kcat is greater by about 40-fold than that of
murine CA IV. These observations represent one of the largest kinetic
differences both qualitatively and quantitatively between a single
isozyme of carbonic anhydrase from different animal species.
The data for kcat describing CO2 hydration catalyzed by H64A mCA IV, although lacking the proton shuttle His64, clearly show the emergence of a second proton shuttle in the enhanced activity at the higher pH range of Fig. 6. Hence, murine CA IV demonstrates at least two pathways for intramolecular proton transfer. Its further study may clarify the properties of multiple proton transfers and clarify the role of such processes in more complex cases of multiple proton pathways recently reported in enzymes (glycinamide ribonucleotide transformylase (35)) and in proton-translocation proteins (cytochrome oxidase (36) and photosynthetic reaction center (37)). Each of these cases has a similarity to our results in that mutation of a single proton-transfer residue leaves considerable activity, indicating that these residues are nonessential when mutated individually and suggesting multiple proton-transfer pathways.
Interconversion of CO2 and HCO3The ratio
kcat/Km contains rate
constants for the catalysis up to the first irreversible step in the
pathway, which in the hydration direction is the release of
HCO3 in Equation 5. Thus,
kcat/Km contains information
on the rate of interconversion of CO2 and
HCO3
but not on the proton-transfer steps by which
the zinc-bound hydroxide is regenerated (Equation 6). The maximal value
of kcat/Km for hydration of
CO2 catalyzed by mCA IV was similar when measured at
25 °C by 18O exchange (3.2 × 107
M
1·s
1; Fig. 3) and by stopped
flow spectrophotometry (5.0 × 107
M
1·s
1; data not shown). The
apparent pKa for this activity was 6.6 (Fig. 3).
Hence, when Wistrand and Knuuttila (7) and Maren et al. (12)
made measurements of
kcat/Km for hydration at pH
near 7.0-7.8 they were close to maximal values; their measurements
were made at 0 to 1 °C and yielded
kcat/Km near 2 × 107 M
1·s
1 for
human and bovine CA IV. These values are to be compared with the
maximal value of kcat/Km for
human CA II of 1.5 × 108
M
1·s
1 at 25 °C (24).
The pH dependence of kcat/Km
for hydration of CO2 catalyzed by mCA IV gave an apparent
pKa of 6.6 ± 0.1 measured by 18O
exchange at chemical equilibrium (Fig. 3); this is a direct indication
of the pKa of the zinc-bound water molecule (38). A
consistent result is the pKa of 6.5 ± 0.2 for the hydrolysis of 4-nitrophenyl acetate catalyzed by mCA IV (Fig. 4).
However, for reasons that are unclear, the apparent
pKa measured from
kcat/Km at steady state gave
a value somewhat larger, 7.3 ± 0.2. The value of the
corresponding pKa for CA II is 6.9 but depends
significantly on ionic strength and sulfate concentration (38). In view
of these similarities, it is interesting that the maximal value of
kcat/Km for the hydrolysis of
4-nitrophenyl acetate catalyzed by mCA IV at 20 ± 1 M1·s
1 is less by a factor of
at least 100 than that measured for CA II, which is as great as 3 × 103 M
1·s
1 (27,
39). The replacement His64
Ala in mCA IV had an effect
on kcat/Km for hydration of
CO2 measured by 18O exchange; the maximal value
is enhanced somewhat and the apparent pKa of
7.3 ± 0.1 is also increased compared with that of the unmodified
mCA IV (Fig. 3). These are small effects relative to those observed for
kcat and RH2O but reflect
the influence of the side chain of residue 64 on the ionization of the
zinc-bound water and the related effect on the rate of interconversion
of CO2 and HCO3
.
We acknowledge the technical assistance of Yanping Zhang and Yang Wang.