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.
