John T. Edsall

Abstract Both human carbonic anhydrases B and C act as esterases on o- and p-nitrophenyl acetates. Enzyme C is the more active of the two for the hydrolysis of p-nitrophenyl acetate, and enzyme B for o-nitrophenyl acetate. The pH-activity curves are sigmoid, the esterase activity being very small below pH 6 and rising to a high level around pH 9; the inflection point lies at pH 7.3 for Enzyme B and at 6.8 for Enzyme C. The Km values are nearly independent of pH in all cases; thus the pH dependence curves appear to reflect the catalytic center activity. The reactions follow Michaelis-Menten kinetics over the range of substrate concentrations studied, but this range is limited to values less than the Km values, because of the limited solubility of the esters. Measurements in a stop-flow apparatus, at times from 10 msec to 2 sec, gave the same kinetic constants as those measured under steady state conditions. There was no evidence of an initial burst of release of nitrophenol. When the reactions were studied under the condition (E0) ≅ (S0) << Km, the process followed first order kinetics until hydrolysis was nearly complete. The data thus gave no evidence for the presence of an acyl intermediate; if such an intermediate exists it must be very rapidly hydrolyzed. Both enzymes are inhibited by monovalent anions, by acetazolamide, and by alcohols. Anion inhibition decreases with increasing pH, and so does the acetazolamide inhibition. The alcohol inhibition is not affected by pH. Enzyme B is somewhat more strongly inhibited by anions, but Enzyme C is much more strongly inhibited by alcohols and by acetazolamide. Only one site seems to be involved in the inhibition by either type of inhibitor. Competition for one site has been demonstrated between the anions and the alcohols. The inhibitions are reversible, and noncompetitive with respect to substrate. Since anion inhibition follows the lyotropic series, the binding site is believed not to be the zinc ion. Binding of anions by Enzyme B, and by zinc-free apoenzyme, has been demonstrated by an increase in pH of an isoionic solution caused by the addition of neutral salts. At low salt concentrations solutions of the apoenzyme show a lower change in pH than these of the holoenzyme, but at higher salt concentrations the values are very similar. Thus in 0.1 m KCl both the enzyme and apoenzyme bind approximately 6 chloride ions.

Abstract Three different methods are described for separating hemoglobins from carbonic anhydrases in hemolysates from human erythrocytes. The preferred method involves adsorption on diethylaminoethyl Sephadex at pH 8.7 followed by selective elution of the carbonic anhydrases. Carbonic anhydrases A, B, and C are subsequently separated from each other on DEAE-Sephadex by elution with 0.05 m Trischloride buffer at pH 8.7. Complete amino acid analyses are reported for carbonic anhydrases B and C, with results in good accord with those of preparations obtained in other laboratories by different procedures. The partial specific volume of carbonic anhydrase B is found to be 0.731 ml per g and its intrinsic viscosity is 2.76 ml per g. Molecular weights from sedimentation, diffusion, and sedimentation equilibrium are close to 28,000 for both enzymes; estimates from the amino acid analyses are 28,730 for Enzyme B, and 30,000 for Enzyme C. The s020, w values are close to 2.75 S for both enzymes. The values for both s and D are lower than others previously reported, by a factor close to 1.2, but the resulting molecular weights are in good agreement with others. Studies of optical rotatory dispersion between 330 and 600 mµ are reported for the native and the acid-denatured carbonic anhydrases B and C, and also for solutions containing the substrate bicarbonate ion and, in other experiments, the inhibitor acetazolamide. The data in all these solvents give a good fit to the simple Drude equation, with λc values that lie below 210 mµ for native Enzyme B and average close to 210 mµ for Enzyme C. Analysis of the data in terms of the Moffitt-Yang and Shechter-Blout equations is also reported, but a computer analysis indicates that the basis for applying the former equation is dubious. There appears to be a small amount of helix (10 to 20%) in the acid-denatured proteins; the complexity of the optical rotatory dispersion spectra of the native proteins makes inferences concerning helix content uncertain (see following paper). Studies on the esterase activity of Enzyme B, with p-nitrophenyl acetate as substrate, are reported. Esterase activity rises with increasing pH between 7 and 9.5. Acetazolamide is a powerful inhibitor of the esterase activity, with Ki = 0.3 µm at pH 7, 0.9 µm at pH 8, and approximately 2.8 µm at pH 9. The inhibition appears to be noncompetitive.