Title: Catalytic Properties of Murine Carbonic Anhydrase IV
Abstract: 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 and18O 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 numberkcat 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. 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 and18O 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 numberkcat 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 (CAs 1The abbreviations used are: CA, carbonic anhydrase; mCA, murine carbonic anhydrase; H64A mCA IV, the mutant of murine carbonic anhydrase IV containing the replacement His64 → Ala; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; Taps, 3[tris(hydroxymethyl)methyl]aminopropanesulfonic acid;D(k), the solvent hydrogen isotope effect onk, (k)H2O/(k)D2O. 1The abbreviations used are: CA, carbonic anhydrase; mCA, murine carbonic anhydrase; H64A mCA IV, the mutant of murine carbonic anhydrase IV containing the replacement His64 → Ala; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; Taps, 3[tris(hydroxymethyl)methyl]aminopropanesulfonic acid;D(k), the solvent hydrogen isotope effect onk, (k)H2O/(k)D2O.) constitute a gene family of at least seven distinct isozymes (1Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Google Scholar) 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%) (2Tashian R.E. BioEssays. 1989; 10: 186-192Google Scholar), they are quite diverse in their cellular distribution, catalytic activity, and physiological function (reviewed in Ref. 3Dodgson S.J. Dodgson S.J. Tashian R.E. Gros G. Carter N.D. The Carbonic Anhydrases. Plenum Publishing Corp., New York1991: 297-306Google Scholar). Among these isozymes, CA IV is the only known membrane-associated form. It was first identified and purified from bovine lung (4Whitney P.L. Briggle T.V. J. Biol. Chem. 1982; 257: 12056-12059Google Scholar), although the presence of a membrane-bound carbonic anhydrase activity had been observed earlier (reviewed in Refs. 5Maren T.H. Ann. N. Y. Acad. Sci. 1980; 341: 246-258Google Scholar and6Wistrand P.J. Ann. N. Y. Acad. Sci. 1984; 429: 195-206Google Scholar). Subsequent purifications from human kidney (7Wistrand P.J. Knuuttila K.-G. Kidney Int. 1989; 35: 851-859Google Scholar, 8Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Google Scholar), human lung (8Zhu X.L. Sly W.S. J. Biol. Chem. 1990; 265: 8795-8801Google Scholar), and lung microsomal membranes from a variety of mammals (9Waheed A. Zhu X.L. Sly W.S. J. Biol. Chem. 1992; 267: 3308-3311Google Scholar) 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. 10Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Google Scholar). Moreover, CA IV is the luminal CA in the proximal tubule of the kidney and is estimated to mediate 85% of renal bicarbonate reabsorption (10Sly W.S. Hu P.Y. Annu. Rev. Biochem. 1995; 64: 375-401Google Scholar). 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 (11Stams T. Nair S.K. Okuyama T. Waheed A. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13589-13594Google Scholar). Two disulfide bridges appear in the structure of human CA IV (11Stams T. Nair S.K. Okuyama T. Waheed A. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13589-13594Google Scholar) 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 (4Whitney P.L. Briggle T.V. J. Biol. Chem. 1982; 257: 12056-12059Google Scholar) 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 (7Wistrand P.J. Knuuttila K.-G. Kidney Int. 1989; 35: 851-859Google Scholar) and bovine CA IV (12Maren T.H. Wynns G.C. Wistrand P.J. Mol. Pharmacol. 1993; 44: 901-905Google Scholar) as a fast isozyme, with a catalytic turnoverkcat of approximately 2 × 105s−1 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 (13Vincent S.H. Silverman D.N. Arch. Biochem. Biophys. 1980; 205: 51-56Google Scholar). Sulfonamides inhibit CA IV, but the average inhibition constant is 17-fold less than for CA II (12Maren T.H. Wynns G.C. Wistrand P.J. Mol. Pharmacol. 1993; 44: 901-905Google Scholar). cDNA clones for human (14Okuyama T. Sato S. Zhu X.L. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1315-1319Google Scholar), rat (15Fleming R.E. Crouch E.C. Ruzicka C.A. Sly W.S. Am. J. Physiol. 1993; 265: L627-L635Google Scholar), and mouse (16Tamai S. Cody L.B. Sly W.S. Biochem. Genet. 1996; 34: 31-43Google Scholar) 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 (17Okuyama T. Waheed A. Kusumoto W. Zhu X.L. Sly W.S. Arch. Biochem. Biophys. 1995; 320: 315-322Google Scholar). Refolding of CA IV inclusion bodies obtained from expression inEscherichia coli 2J. D. Hurt, C. K. Tu, and P. J. Laipis, in preparation. 2J. D. Hurt, C. K. Tu, and P. J. Laipis, in preparation. 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 inE. coli strain BL21(DE3)pLysS using the pET31 T7 expression vector described by Tanhauser et al. (18Tanhauser S.M. Jewell D.A. Tu C.K. Silverman D.N. Laipis P.J. Gene. 1992; 117: 113-117Google Scholar). The mutant H64A mCA IV was constructed using a mutating oligonucleotide (18Tanhauser S.M. Jewell D.A. Tu C.K. Silverman D.N. Laipis P.J. Gene. 1992; 117: 113-117Google Scholar, 19Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Google Scholar) 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 (20Tu C.K. Thomas H.G. Wynns G.C. Silverman D.N. J. Biol. Chem. 1986; 261: 10100-10103Google Scholar). 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. The catalyzed and uncatalyzed rates of 18O exchange from species of CO2 into water and the rates of exchange of 18O between12C-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 Equation2, B− is a buffer in solution and/or an amino acid side chain in the enzyme. HOCO18O−+EZnH2O⇆EZn18OH−+CO2+H2OEquation 1 EZn18OH−+BH⇆EZn18OH2+B−⇆H2OEquation 2 EZnH2O+H218O+B−This method has been described by Silverman (21Silverman D.N. Methods Enzymol. 1982; 87: 732-752Google Scholar) and is capable of determining two rates in the catalytic pathway, as described below. The first is R1, the rate of interconversion of CO2 and HCO3− at chemical equilibrium. Equation 3 expresses the substrate dependence ofR1. Rl/[E]=kcatex[S]/(KeffS+[S])Equation 3 Here [E] is the total enzyme concentration,kcatex is a rate constant for maximal HCO3− to CO2 interconversion, [S] is the substrate concentration of HCO3− and/or CO2, andKeffS is an apparent substrate binding constant (22Simonsson I. Jonsson B.-H. Lindskog S. Eur. J. Biochem. 1979; 93: 409-417Google Scholar). This equation, when applied to the data for varying substrate concentration or to measurement ofR1 when [S] ≪KeffS, can determine the values ofkcatex/KeffS. The second rate determined by this method isRH2O, 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 ofRH2O can be interpreted in terms of the rate constant from a predominant proton donor group to the zinc-bound hydroxide according to Equation 4 (23Silverman D.N. Tu C.K. Chen X. Tanhauser S.M. Kresge A.J. Laipis P.J. Biochemistry. 1993; 32: 10757-10762Google Scholar), in whichkB 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. RH2O/[E]=kB/{(1+KB/[H+])(1+[H+]/KE)}Equation 4 Measurements of the rate of distribution of 18O were determined using an Extrel EMX-200 mass spectrometer and a membrane inlet permeable to dissolved gases (21Silverman D.N. Methods Enzymol. 1982; 87: 732-752Google Scholar). Solutions contained 5 μm EDTA (except for measurement of Cu2+inhibition), and the total ionic strength of solution was maintained at 0.2 m by the addition of Na2SO4. Unless indicated otherwise, experiments were carried out in the absence of buffers, which were not needed to maintain pH since these experiments were carried out at chemical equilibrium. 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 (24Khalifah R.G. J. Biol. Chem. 1971; 246: 2561-2573Google Scholar). 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 0.1m by the addition of the appropriate amount of Na2SO4. Solutions contained 4 μmEDTA. 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 constantskcat andkcat/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 inkcat andkcat/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 × 103m−1·cm−1 (25Verpoorte J.A. Mehta S. Edsall J.T. J. Biol. Chem. 1967; 242: 4221-4229Google Scholar). 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 obtainedkcat/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 (18Tanhauser S.M. Jewell D.A. Tu C.K. Silverman D.N. Laipis P.J. Gene. 1992; 117: 113-117Google Scholar). An alignment of the full-length murine CA IV protein sequence with that of human CA II is shown in Fig. 1. The twoarrows 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 (9Waheed A. Zhu X.L. Sly W.S. J. Biol. Chem. 1992; 267: 3308-3311Google Scholar). 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 (9Waheed A. Zhu X.L. Sly W.S. J. Biol. Chem. 1992; 267: 3308-3311Google Scholar, 15Fleming R.E. Crouch E.C. Ruzicka C.A. Sly W.S. Am. J. Physiol. 1993; 265: L627-L635Google Scholar). 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 (17Okuyama T. Waheed A. Kusumoto W. Zhu X.L. Sly W.S. Arch. Biochem. Biophys. 1995; 320: 315-322Google Scholar). The form of murine CA IV examined here is C terminally truncated two amino acids beyond the corresponding serine in the human sequence (17Okuyama T. Waheed A. Kusumoto W. Zhu X.L. Sly W.S. Arch. Biochem. Biophys. 1995; 320: 315-322Google Scholar). 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 (14Okuyama T. Sato S. Zhu X.L. Waheed A. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1315-1319Google Scholar). 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 (11Stams T. Nair S.K. Okuyama T. Waheed A. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13589-13594Google Scholar). 3D. W. Christianson, personal communication. We anticipate that the presence of glutamate at position 12 has no effect on the catalysis by murine CA IV. The rate constantR1/[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 ofR1/[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 CO2species, 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 ofkcatex in Equation 3. However, we were able to obtainkcatex/KeffSfrom 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 ratiokcatex/KeffCO2(for S = CO2) obtained by 18O exchange is in theory and in practice equivalent tokcat/Km for hydration of CO2 obtained in steady-state measurements (22Simonsson I. Jonsson B.-H. Lindskog S. Eur. J. Biochem. 1979; 93: 409-417Google Scholar). The18O 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 ofkcat/Km of 3.2 ± 0.1 × 107m−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 by18O 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 × 107m−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 wasD(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 ofkcat/Km of 6.3 ± 0.2 × 107m−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 ± 1m−1·s−1 with an apparent pKa of 6.5 ± 0.2 (Fig. 4). The rate constant RH2O/[E] for the proton transfer-dependent release of18O-labeled water from the active site (Equation 2) was measured by mass spectrometry in the absence of buffers; the values ofRH2O/[E] as a function of pH are shown in Fig. 5.RH2O/[E] was evaluated by Equation4 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 × 106s−1) 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 ofkcat/Km for hydration of CO2 and for ester hydrolysis. The solvent hydrogen isotope effect on kB wasD(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 apparentKmbuffer of 80 ± 20 mmand a maximal value of 1.9 ± 0.2 × 105s−1 at pH 7.2 (data not shown). In contrast, the value ofRH2O/[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 CO2catalyzed by mCA IV and measured at steady state had a maximal value at high pH of kcat = 1.1 ± 0.1 × 106 s−1 (Fig. 6). This maximal value of kcat is very similar to that ofkB 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). 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 blockedkcat/Km andRH2O 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/Kmdetermined 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 (11Stams T. Nair S.K. Okuyama T. Waheed A. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13589-13594Google Scholar). 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. CO2+EZnOH−+H2O⇄HCO3−+EZnH2OEquation 5 His64EZnH2O+B−⇄H+His64EZnOH−+B−⇄Equation 6 His64EZnOH−+BHHere BH represents buffer in solution and/or a possible proton shuttle of the enzyme. This scheme of Equations 5 and 6 has been supported by considerable evidence for the mechanism of CA II (26Silverman D.N. Lindskog S. Acc. Chem. Res. 1988; 21: 30-36Google Scholar), and its use for mCA IV is consistent with a number of features of this work: a solvent hydrogen isotope effect near unity,D(kcat/Km) = 0.83 ± 0.11, indicating the lack of proton transfer in Equation5; a solvent hydrogen isotope effect onRH2O/[E] of 1.9 ± 0.4 consistent with primary proton transfer in Equation 6; and an effect of buffer imidazole on kcat but not onkcat/Km. 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. The 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 (27Steiner H. Jonsson B.-H. Lindskog S. Eur. J. Biochem. 1975; 59: 253-259Google Scholar, 28Rowlett R.S. J. Protein Chem. 1984; 3: 369-393Google Scholar). The similar values ofkcat for isozymes II and IV as well as the presence and similar conformation of His64 in both of these isozymes (11Stams T. Nair S.K. Okuyama T. Waheed A. Sly W.S. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13589-13594Google Scholar) 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 s−1 (Fig. 6), quite close in magnitude to the kcat value of 1.4 × 106 s−1 measured under comparable conditions for human CA II (24Khalifah R.G. J. Biol. Chem. 1971; 246: 2561-2573Google Scholar). Further support for the role of His64 in CA IV comes from18O-exchange studies. The rate constant for the release of18O-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 (29Tu C.K. Silverman D.N. Forsman C. Jonsson B.H. Lindskog S. Biochemistry. 1989; 28: 7913-7918Google Scholar). 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. These18O-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 His64in human CA II, which yields a value of pKa = 7.1 (30Campbell I.D. Lindskog S. White A.I. J. Mol. Biol. 1975; 98: 597-614Google Scholar). These same 18O-exchange data of Fig. 5 give a maximal rate constant for intramolecular proton transfer from His64to the zinc-bound hydroxide molecule of kB = 1.4 ± 0.2 × 106 s−1, 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 furt