Catalytic Properties of Murine Carbonic Anhydrase IV (1997)

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 exchange between CO2 and water measured by The a efficient enzyme but with that are the studied isozymes of the human and carbonic the maximal turnover to be at least suggesting that than proton shuttle is in the catalytic of these is a shuttle with a pKa near is identified as His64 by are in the of a of suggesting the role of multiple proton in catalysis and A murine lung carbonic anhydrase IV cDNA was and a of the to was expressed coli using the expression described by Silverman D.N. P.J. 1992; Scholar). The mutant H64A mCA IV was using a Silverman D.N. P.J. 1992; Scholar, K. J. Scholar) and by The were and mCA inclusion bodies were in and an active was using a of and exchange Wynns G.C. Silverman D.N. J. Biol. Chem. Scholar). The of the enzyme was estimated at than by The of active carbonic anhydrase was by with the carbonic anhydrase the catalyzed exchange between CO2 and than of the enzyme was active, that nearly of the purified mCA IV had been the active The catalyzed and of exchange from of CO2 water and the of exchange of and of CO2 were measured at using a 1 and 2 the catalytic for the exchange of from bicarbonate to water. In is a in solution an amino acid in the 1 2 has been described by Silverman D.N. 1982; Scholar) and is of in the catalytic as described The first is the of of CO2 and HCO3− at the is the enzyme is a constant for maximal HCO3− to CO2 is the of HCO3− is an constant S. J. Biochem. 93: Scholar). to the for or to the The second by this the of from the enzyme of water with A proton from a the zinc-bound to zinc-bound with water. The is by the solvent water. The be in of the constant from a proton to the zinc-bound to D.N. X. P.J. 1993; Scholar), in is the constant for proton transfer to the zinc-bound is the constant for the and is the constant of the zinc-bound water molecule. Measurements of the of distribution of were using an and a membrane to D.N. 1982; Scholar). for of and the of solution was at by the of were in the of were not to pH these were at A stopped flow was used to initial velocities of the hydration of this catalysis protons as as we measured the initial of hydration by the of a pH J. Biol. Chem. Scholar). CO2 were by CO2 water at with were used to CO2 from to The pKa of the and the observed were as with with with with and with The was and the for was at by the of the of The of to of the first to of the was used to initial The were and the were by least The of for hydration of CO2 were below at pH and a maximal near at pH we were to of CO2 over most of the pH range The were at The catalysis by mCA IV of the of was measured by the in at to the of and the using the × S. J. Biol. Chem. Scholar). the catalysis of this by mCA IV was we the and from the in the presence of to inhibit mCA IV. The was the of the to the at the active velocities were the in the to The of for catalysis was to the of a we were limited to catalytic that were first in the from we A murine CA IV cDNA in structure to human CA II was a and protein expressed in coli Silverman D.N. P.J. 1992; Scholar). of the murine CA IV protein with that of human CA II is in The in 1 the initial and amino of the expressed mCA IV used in this human and rat CA IV from lung have been to be by and amino to the membrane (9Waheed A. Zhu X.L. Sly W.S. J. Biol. Chem. 1992; 267: 3308-3311Google Scholar). The murine CA IV was to that of the rat the first amino and the a The in the mouse that this to the second in the mature rat protein (9Waheed A. Zhu X.L. Sly W.S. J. Biol. Chem. 1992; 267: 3308-3311Google Scholar, R.E. Crouch E.C. Ruzicka C.A. Sly W.S. Am. J. Physiol. 1993; 265: L627-L635Google Scholar). CA IV is C and to a by this membrane be enzyme activity, and a active form of human CA IV be expressed the C to to in and to a (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 is C amino the in the human (17Okuyama T. Waheed A. Kusumoto W. Zhu X.L. Sly W.S. Arch. Biochem. Biophys. 1995; 320: 315-322Google Scholar). of the murine CA IV expression this a at to a is a 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). in a region of human CA IV, is a that solvent and has with other 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). W. that the presence of at has effect the catalysis by murine CA IV. The for the of CO2 and HCO3− at catalyzed by mCA IV was measured as a function of of of with at pH in The the of the + was nearly with of example, the in the of 2 is with a of of where both CO2 and this of the of CO2 and we were not to a in we were to the of as the in 2 or from studies with + The for the hydration of CO2 was by by of the exchange of between CO2 and water and by stopped flow of the initial of CO2 is an distribution of enzyme in the obtained by exchange is in and in for hydration of CO2 obtained in S. J. Biochem. 93: Scholar). was at a of of CO2 in the to for + for catalysis by mCA IV a maximal of × with an pKa for the catalysis of 6.6 The presence of to in solution had effect measured exchange for in The of for hydration of CO2 by stopped flow with less not the of these a maximal of of × with an pKa of The solvent hydrogen isotope effect for mCA IV by exchange measured at pH and in containing The exchange studies to catalysis by the mutant H64A mCA IV a maximal of × with an pKa of The of the pKa for catalysis by mCA IV was confirmed by of the catalytic of The of mCA IV in this catalysis was that we were catalysis at the active site as is in hydration of we that of the of catalytic of that was in the presence of a and of mCA IV. The maximal of for this was with an pKa of The constant for the proton water from the active site was measured by in the of the as a function of pH are in was by to a constant for proton transfer to the zinc-bound as as of pKa for the proton and for the zinc-bound water molecule. was by least to the for catalysis by mCA IV to the constant for proton transfer × with pKa for the proton and pKa 6.6 for the zinc-bound water molecule. is in with the for the zinc-bound water molecule obtained by for hydration of CO2 and for The solvent hydrogen isotope effect catalyzed by H64A mCA IV was less than that for the enzyme by close to 100-fold The of catalyzed by H64A mCA IV was enhanced than and in a by the of with an of a maximal of × 105s−1 at pH not In the catalyzed by mCA IV was enhanced by of at the of the The catalytic turnover for the hydration of by mCA IV and measured at had a maximal at high pH of kcat 1.1 × 106 s−1 maximal of kcat is to that from The pH of kcat could not be to a single and shows the of or an inhibition from other in the region of pH between and 9 collected in the presence of this effect kcat suggesting that is in to proton transfer in the active The turnover kcat for CO2 hydration catalyzed by H64A mCA IV was less than kcat for the mCA IV The the inhibition of exchange between CO2 and water catalyzed by mCA IV, were from collected at 1 for of these only with a of the and of by exchange was not by as as In this study we the catalytic of murine CA IV an and an enzyme to resemble both the mature form of CA IV and CA II, a studied and efficient of carbonic amino acid of this murine CA IV with that of human CA II is in The identity of in to the and in hydrogen to the zinc-bound water molecule that are in of the carbonic Moreover, the crystal of human CA IV and human CA II in the region of their active are (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). these the catalytic of murine CA IV be by to II as described in and in solution a proton shuttle of the of and has been by considerable for the mechanism of CA II D.N. S. Chem. Scholar), and its for mCA IV is with a of of this a solvent hydrogen isotope effect near the of proton transfer in a solvent hydrogen isotope effect of with proton transfer in and an effect of kcat but not these between CA II and CA IV, catalysis by murine CA IV the isozymes of carbonic anhydrase the of that its catalysis multiple proton transfer as as the role of His64 in the proton shuttle is The maximal turnover for CO2 hydration catalyzed by CA II has been in to be in by the proton transfer from the zinc-bound water molecule to His64 S. J. Biochem. Scholar, J. Chem. 1984; Scholar). The for isozymes II and IV as as the presence and 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) that the proton transfer to the maximal of CA IV. The maximal turnover kcat for the hydration of CO2 catalyzed by mCA IV at was observed to be 1.1 × 106 s−1 quite close in magnitude to the kcat of × 106 s−1 measured for human CA II J. Biol. Chem. Scholar). for the role of His64 in CA IV The constant for the water from mCA in a pH to that of CA II Silverman D.N. C. S. 1989; Scholar). is by the of the presence of the zinc-bound and the presence of a second that a proton to the zinc-bound as the of the pKa of 6.6 for the of the zinc-bound water and the pKa of for the second this is close to that by the of the proton of the proton of human CA II, a of pKa S. J. Mol. Biol. Scholar). of a maximal constant for proton transfer from the zinc-bound molecule of × 106 with the maximal of is by this proton these be nearly identical for proton in the hydration and is the of the of the pKa for and nearly identical at pKa The role of His64 as proton in CA IV is by the inhibition of catalysis by and by the of kcat for the mutant H64A mCA IV. The for the mCA IV showed inhibition of × with effect the of of CO2 and to a of are with an inhibition of the with effect the function of the zinc-bound in the first of catalysis that was obtained with CA II, in was by with an of × and by with an of × with effect Wynns G.C. Silverman D.N. J. Biol. Chem. Scholar). was as an inhibition of the function of His64 as a proton shuttle and was confirmed by the crystal structure of the II a of with to and to of the of His64 A. C. W. Scholar). The inhibition of CA IV and CA II by is for the role of proton transfer by CA IV. Moreover, the near 2 × s−1 for CA IV in the presence of is close to the for the mutant H64A CA IV in His64 is by and is close to the of for H64A CA II Silverman D.N. C. S. 1989; Scholar), suggesting that this of has nearly the proton transfer role of a shuttle The remaining activity is to other less efficient of the protein or to water in the active inhibition suggesting a proton shuttle role for His64 were by the lower for hydration catalyzed by the mutant H64A mCA IV and of these constants are by proton transfer from or to the zinc-bound water or S. J. Biochem. Scholar, Silverman D.N. C. S. 1989; Scholar) in CA is confirmed by the of for H64A mCA IV by of a by the from solution Although the maximal of kcat for hydration are for mCA IV and CA II, is a the pH of CA II be described by a single J. Biol. Chem. Scholar), that of the proton but the pH for mCA IV is the of at least is that the in the pH profile for kcat was not in the observation is most likely to was at in the of did not a proton transfer to and not be as by limited of His64 as kcat in the proton transfer to in solution. observation is that to be an proton with pKa 9 that to an of kcat for H64A mCA IV and for the mCA IV as A observation was for murine CA Christianson D.W. P.J. Silverman D.N. 1996; 35: Scholar). this was not observed in the the transfer of protons from a with pKa 9 to the zinc-bound the acid of has pKa near 6.6 a that the to proton transfer from of these is His64 as a proton as described and the second is at least other proton shuttle of pKa near 9 that has not been identified but that is to that observed in CA Christianson D.W. P.J. Silverman D.N. 1996; 35: Scholar). The by the presence of that the in the pH profile of kcat for mCA IV near pH is to of the proton mCA IV has the to with an proton a that that the function of the proton shuttle His64 in mCA IV is for the pH profile of mCA IV be the presence of at human CA II has an at this the proton shuttle function of His64 in a to that of in CA Christianson D.W. P.J. Silverman D.N. 1996; 35: Scholar). in CA II C.A. 1996; 35: Scholar) found effect of the replacement → the function of the mechanism It is that murine CA IV has at human CA II has at this in this murine CA IV has a that could the role of His64 as a proton S. Waheed A. Cody L.B. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 93: Scholar) have reported with the of in the catalysis by CA the replacement → in bovine CA IV catalytic activity measured in a near and the replacement → in murine CA IV role of the in the pH profile for kcat obtained for murine CA IV in this and that of Waheed A. Okuyama T. Sly W.S. C.A. Scholar) for human CA IV. observed a pH profile catalyzed by human CA IV that is with a single at physiological pH this is by than that of murine CA IV. of the kinetic both and between a single of carbonic anhydrase from The for kcat catalyzed by H64A mCA IV, although the proton shuttle the of a second proton shuttle in the enhanced activity at the pH range of murine CA IV at least pathways for proton study the of multiple proton and the role of in of multiple proton pathways reported in S.J. 1996; 35: and in P. Y. P. S. 1996; 35: Scholar) and 1995; 34: of these has a similarity to in that of a single considerable activity, that these are and suggesting multiple The constants for the catalysis to the first in the in the hydration is the of HCO3− in the of of CO2 and HCO3− but not the by the zinc-bound is The maximal of for hydration of CO2 catalyzed by mCA IV was measured at by exchange × and by stopped flow spectrophotometry × not The pKa for this activity was 6.6 Wistrand and Knuuttila (7Wistrand P.J. Knuuttila K.-G. Kidney Int. 1989; 35: 851-859Google Scholar) and (12Maren T.H. Wynns G.C. Wistrand P.J. Mol. Pharmacol. 1993; 44: 901-905Google Scholar) for hydration at pH near they were close to maximal their were at 0 to 1 and near 2 × for human and bovine CA IV. are to be with the maximal of for human CA II of × at J. Biol. Chem. Scholar). The pH of hydration of CO2 catalyzed by mCA IV an pKa of 6.6 measured by exchange at this is a of the pKa of the zinc-bound water molecule S. J. Biochem. 1982; Scholar). A is the pKa of for the of catalyzed by mCA IV for that are the pKa measured at a The of the pKa for CA II is but significantly and S. J. Biochem. 1982; Scholar). In of these is that the maximal for the of catalyzed by mCA IV at is less by a of at least than that measured for CA II, is as as × S. J. Biochem. Y. T. Scholar). The replacement His64 → in mCA IV had an effect for hydration of CO2 measured by the maximal is enhanced and the pKa of is with that of the mCA IV are to those observed and but the of the of the of the zinc-bound water and the effect the of of CO2 and the of and