Carbonic anhydrase in human platelets.

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.     

Hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.     

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.     

A search for the function of human carbonic anhydrase B.

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.     

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.     

A comparison of the kinetic properties of native bovine muscle carbonic anhydrase and an activated derivative with modified thiol groups

Steady-state and equilibrium kinetic properties of native bovine carbonic anhydrase III (carbonate hydrolyase, EC 4.2.1.1) and a derivative modified with methyl methanethiosulfonate were investigated. The modified enzyme has a markedly increased CO2 hydration activity compared to the native form with a 3-times higher value of kcat and a 6-10-times higher value of kcat/Km. Qualitatively, the activated enzyme shows the same kinetic behavior as native isoenzyme III. This is reflected in similar pH dependences of the kinetic parameters for CO2 hydration, similar solvent hydrogen isotope effects on these parameters, similar deviations from Michaelis-Menten kinetics for the HCO3- dehydration reaction, and similar behavior of the kinetics of CO2/HCO3- exchange at chemical equilibrium as measured by a 13C-NMR magnetization transfer technique. It is concluded that the conversion of -SH groups to -S-S-CH3 moieties does not change the catalytic mechanism, but leads to an increased rate of CO2/HCO3- interconversion as well as to an increased rate of proton transfer between the active site and the reaction medium.     

Activation and inhibition of bovine carbonic anhydrase III by dianions

We have found that many dianionic species, at millimolar concentrations, significantly activate or inhibit the bovine carbonic anhydrase III-catalyzed hydration of CO2. Dianionic species such as HPO2-4 and SO2-3, with pKb values near 7, are activators, whereas weakly basis species such as SO2-4 act as inhibitors. Both activation and inhibition are partial hyperbolic in nature and do not appear to compete with monoanionic linear inhibitors like N-3. Our kinetic data are consistent with a formal mechanism of action for carbonic anhydrase III that is directly analogous to that of carbonic anhydrase II, in which Lys-64 of carbonic anhydrase III can act as an intramolecular H+ transfer group during CO2 hydration. Our data suggest that dianionic inhibitors depress the rate of H+ transfer during turnover by stabilizing the protonated form of Lys-64. We postulate that dianionic activators enhance the rate of a rate-limiting H+ transfer step in the mechanism, probably by acting directly as H+ acceptors.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Carbonic anhydrase assay: strong inhibition of the leaf enzyme by CO2 in certain buffers

Apparent carbonic anhydrase activity in leaf extracts, measured as the rate of H+ production associated with the CO2 hydration reaction, varied by as much as 25-fold when the assay buffer was varied. Highest activities were usually recorded in barbitone buffer, with lower activities in imidazole, Tricine, Hepes, Tris, and phosphate buffers. The greatest differences were observed with the enzyme isolated from leaves of the monocotyledonous plants Zea mays (maize) and Triticum aestivum (wheat). Smaller differences were observed with carbonic anhydrase from dicotyledonous species and there was no effect on the erythrocyte enzyme. Leaf carbonic anhydrase activity measured by the mass spectrometric procedure was unaffected by varying the assay buffer. The low activity in certain buffers observed with the former assay system was found to be due to inhibition of the enzyme-catalyzed reaction by higher concentrations of CO2. Carbonic anhydrase from some sources was also strongly inhibited by certain inorganic and organic anions.     

Purification and characterization of carbonic anhydrase from the saliva of the rat

Carbonic anhydrase purified from the saliva of the rat had kinetic properties identical with those of carbonic anhydrase II from rat red cells, but its molecular properties were distinctly different from the type II isozyme. Kinetic parameters were measured under steady state conditions by stopped-flow spectrophotometry and under equilibrium conditions by an 18O exchange method. The turnover number kcat for hydration of CO2 was 6.5 X 10(4) s-1 and the Michaelis constant was 4.2 mM at pH 7.5 and 25 degrees C, values which are equal to the steady state constants for red cell carbonic anhydrase II from the rat. Inhibition of the salivary isozyme by sulfanilamide (Ki = 3.7 microM) was nearly as efficient as inhibition of the erythrocyte isozyme II (Ki = 1.1 microM). The molecular weight for the salivary isozyme was 46,000 and the isoelectric point was 5.5. Salivary carbonic anhydrase had high mannose oligosaccharide components as measured by concanavalin A binding. The amino acid composition for the salivary isozyme was not similar to rat type II, but it was similar to that reported for membrane-bound carbonic anhydrase from bovine lung (Whitney, P.L., and Briggle, T.V. (1982) J. Biol. Chem. 257, 12056-12059). These observations suggest to us that salivary carbonic anhydrase is a secretory product.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.     

Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2

We have determined the activation parameters of kcat and kcat/Km for the carbonic anhydrase II-catalyzed hydration of CO2. The enthalpy and entropy of activation for kcat is 7860 +/- 120 cal mol-1 and -3.99 +/- 0.42 cal mol-1 K-1, respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kcat for the human enzyme was further decomposed into partially compensating electrostatic(es) (delta S*es = +15.1 cal mol-1 K-1) and nonelectrostatic(nes) (delta S*nes = -19.1 cal mol-1 K-1) terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase II-catalyzed CO2 hydration show that 82% of the temperature effect on kcat can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition state of the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubban and Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).     

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.     

Fine tuning of the catalytic properties of carbonic anhydrase. Studies of a Thr200----His variant of human isoenzyme II

The active sites of carbonic anhydrases I contain a unique histidine residue at sequence position 200. To test the hypothesis that His200 is essential for the isoenzyme-specific catalytic and inhibitor-binding properties of carbonic anhydrases I, a variant of human carbonic anhydrase II, having His200 for Thr200, was prepared by oligonucleotide-directed mutagenesis. The variant has a circular dichroic spectrum that is indistinguishable from that of the parent enzyme. The kinetics of CO2 hydration and HCO3- dehydration has been investigated. The results show that the amino acid substitution has led to changes of catalytic parameters as well as Ki values for anion inhibition in the expected directions towards the values for isoenzyme I. However, the maximal 4-nitrophenyl acetate hydrolase activity of the variant is higher than for any naturally occurring carbonic anhydrase studied so far. A detailed analysis of the kinetic observations suggests that the modification has resulted in a change of the step that limits the maximal rate of CO2 hydration at saturating buffer concentrations. This rate-limiting step is an intramolecular proton transfer in unmodified isoenzyme II and, presumably, HCO3- dissociation in the variant and in human isoenzyme I. A free-energy profile for the dominating pathway of CO2 hydration at high pH was constructed. The results suggest that the major effect of His200 is a stabilization of the enzyme-HCO3- complex by about 7.5 kJ/mol (variant) and 6.1 kJ/mol (human isoenzyme I) relative to unmodified isoenzyme II, while proton transfer between the metal site and the reaction medium is only marginally affected by the amino acid replacement.