Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

Localization of the multifunctional protein CAD in astrocytes of rodent brain.

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.     

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.     

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.     

An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted ratfish (Hydrolagus colliei)

Separated plasma and whole blood non-bicarbonate buffering capacities, together with plasma and gill carbonic anhydrase activities and endogenous plasma carbonic anhydrase inhibitor activity were investigated in three species of fish: the brown bullhead (Ameirus nebulosus), a teleost; the longnose skate (Raja rhina), an elasmobranch; and the spotted ratfish (Hydrolagus colliei), a chimaeran. The objective was to test the hypothesis that species possessing gill membrane-bound carbonic anhydrase and/or plasma carbonic anhydrase activity would also exhibit high plasma nonbicarbonate buffering capacity relative to whole blood non-bicarbonate buffering capacity and would lack an endogenous plasma carbonic anhydrase inhibitor. Separated plasma non-bicarbonate buffering capacity constituted > or = 40% of whole-blood buffering in all three species. In addition, all species lacked an endogenous plasma carbonic anhydrase inhibitor. Separated plasma from skate and ratfish contained carbonic anhydrase activity, whereas bullhead plasma did not. Examination of the subcellular distribution and characteristics of branchial carbonic anhydrase activity revealed that the majority of branchial carbonic anhydrase activity originated from the cytoplasmic fraction in all species, with only 3-5% being associated with a microsomal fraction. The microsomal carbonic anhydrase activity of bullhead and ratfish was significantly reduced by washing, indicating the presence of carbonic anhydrase activity that was not integrally associated with the membrane pellet, microsomal carbonic anhydrase activity in skate was unaffected by washing. In addition, microsomal carbonic anhydrase activity from skate and ratfish but not bullhead gills was released to a significant extent from its membrane association by treatment with phosphatidylinositol-specific phospholipase C. The results obtained for skate are consistent with published data for dogfish, suggesting that the possession of branchial membrane-bound carbonic anhydrase activity may be a generalised elasmobranch characteristic. Ratfish, which also belong to the class Chondrichthyes, exhibited a similar pattern. Unlike skate and ratfish, bullhead exhibited high plasma non-bicarbonate buffering capacity and lacked an endogenous carbonic anhydrase inhibitor in the absence of plasma and gill membrane-bound carbonic anhydrase activities.     

The effects of the inhibition of the renal carbonic anhydrase on the blood acid-base status in hypercapnic rats

Arterial blood acid-base status was measured in unanesthetized rats treated with benzolamide (a selective renal carbonic anhydrase inhibitor). These measurements were carried out in rats exposed to different levels of CO2 in air (0-10% CO2) for periods of up to 6 hr. In untreated rats the whole body buffer value showed a continuous increase and after 6 hr of exposure to hypercapnia its value was twice that measured initially. On the other hand, the whole body buffer value of benzolamide treated rats did not change during the 6 hr of exposure to hypercapnia. The whole body buffer value of normal rats, measured after 6 hr of hypercapnia is similar to that reported for chronic (3-5 days) hypercapnia in the normal dog. The whole body buffer value in benzolamide treated rats was similar to that reported for the normal dog and man, during acute CO2 exposures. It is suggested that mechanisms involving the renal carbonic anhydrase are responsible for the significant, rapid changes in the whole body buffer value that take place during the initial phase of acute exposure to CO2 in the rat. This may represent a mechanism of adaptation to burrow hypercapnic conditions.     

Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.     

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span

Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.     

Chemical modification of carbonic anhydrase II with acrolein

We have reacted acrolein with human carbonic anhydrase II using conditions reported to result in maximal formylethylation of exposed histidine and lysine residues (Pocker, Y., and Janji?, N. (1988) J. Biol. Chem. 263, 6169-6176). Pocker and Janji? proposed that the decrease by 95-98% in the steady-state turnover number for the hydration of CO2 caused by this chemical modification is due predominantly to the alkylation of one residue, the imidazole side chain of histidine 64. We measured the rate of 18O exchange between CO2 and water catalyzed by these enzymes at chemical equilibrium using membrane inlet mass spectrometry. The catalyzed rate of interconversion of CO2 and HCO3- at chemical equilibrium was the same for the acrolein-modified and the unmodified carbonic anhydrases, but the rate of release of 18O-labeled water from the active site had decreased by as much as 85% for the acrolein-modified enzyme. The 18O-exchange kinetics catalyzed by the acrolein-modified carbonic anhydrase II was similar to that catalyzed by a mutant human carbonic anhydrase II in which histidine at residue 64 was replaced with alanine. Moreover, modification of this mutant carbonic anhydrase II with acrolein did not alter to a significant extent its 18O-exchange pattern. These results support the proposal of Pocker and Janji? and the suggested role of histidine 64 in carbonic anhydrase II as a proton shuttle residue that transfers a proton from zinc-bound water to buffer in solution.     

Further studies on the effect of aldosterone on Mg2+-HCO3(-)-ATPase and carbonic anhydrase from rat intestinal mucosa

The main purpose of this study is to elucidate the effect of adrenocorticoids on Mg2+-HCO3(-)-ATPase and carbonic anhydrase which are thought to be related to anion transport in mammalian intestinal mucosa and renal tubulus. Rat duodenal mucosa, large intestinal mucosa and kidney cortex were excised and homogenized with mannitol-Tris buffer (pH 7.1) and brush border fraction and cytosol were obtained by a differential fractionation procedure. Brush border Mg2+-HCO3(-)-ATPase and cytosol carbonic anhydrase activities in the duodenal mucosa decreased to 70% and 37% of normal values, respectively 5-11 days after adrenalectomy. Adrenalectomy also decreased significantly both enzyme activities in large intestinal mucosa; on the other hand, renal enzyme activities did not change. Four hours after a single injection of 20-80 micrograms/kg of aldosterone, ip, to adrenalectomized rats, Mg2+-HCO3(-)-ATPase and carbonic anhydrase activities in duodenal mucosa increased gradually to normal or near normal in dose-dependent fashion. Both enzyme activities in large intestinal mucosa were also increased by a larger dose of aldosterone. Again, renal enzyme activities were not affected by any dose of aldosterone. In contrast, corticosterone (1 mg and 4 mg/kg) and dexamethasone (50 micrograms 200 micrograms/kg) had no replacement effect on enzyme activities in all organs. These results showed that the mineralocorticoid, but not glucocorticoids, is a regulator of the enzyme activity of Mg2+-HCO3(-)-ATPase and carbonic anhydrase from intestinal mucosa. The true mechanisms by which both enzymes are activated by aldosterone are not clear at present.     

Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii

A new isoenzyme of carbonic anhydrase has been isolated and purified from Chlamydomonas reinhardtii. This carbonic anhydrase is composed of two nonidentical subunits with apparent molecular masses of 39 and 4.5 kDa and is located in the periplasmic space. This is the second periplasmic carbonic anhydrase found in C. reinhardtii. Two genes, CAH1 and CAH2, which code for carbonic anhydrase, have been recently described by Fujiwara et al. (Fujiwara, S., Fukuzawa, H., Tachiki, A., and Miyachi, S. (1990) Proc. Natl. Acad, Sci. U.S.A. 87, 9779-9783). The CAH1 gene codes for a periplasmic carbonic anhydrase which is induced under low CO2 conditions and is well characterized. The carbonic anhydrase characterized in this report was isolated from a mutant that is unable to synthesize the CAH1 gene product. Amino acid sequencing demonstrates that this newly isolated carbonic anhydrase is the CAH2 gene product. This is the first report of another functional carbonic anhydrase in C. reinhardtii.     

Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.