CATALYTIC VERSATILITY OF ERYTHROCYTE CARBONIC ANHYDRASE .4. KINETIC STUDIES OF ENZYME-CATALYZED HYDROLYSES OF P-NITROPHENYL ESTERS

Kinetic studies on the esterase activity of bovine carbonic anhydrase with p-nitrophenyl propionate, n-butyrate, isobutyrate, n-valerate, isovalerate, n-caproate, and trimethyl acetate as substrates are reported. Esterase activity rises continuously but non-uniformly with increasing pH between 4.0 and 10.9. Below pH 9.0 the profiles for propionate, n-butyrate, isobutyrate, and isovalerate are quite similar to that previously obtained for p-nitrophenyl acetate: they are sigmoidal with an inflection occurring around pH 7.3. In contrast, the two longer substrates, p-nitrophenyl n-valerate and p-nitrophenyl n-caproate, exhibit an “abnormal” behavior, (i) Their rate profiles show inflections at pH 6.2 and 5.7, respectively, rather than at 7.3, and (ii) their Ki values for the specific inhibitor acetazolamide are 103 times larger than those found with p-nitrophenyl acetate and propionate as substrates. The sterically hindered trimethyl acetate ester is subject to little or no enzymatic catalysis below pH 8.5. The enzymatic hydrolyses of all the esters reported in this paper exhibit a second inflection, of much greater magnitude around pH 10.5; this dramatic increase in enzymatic activity at high pH has not been reported in any previous kinetic study pertaining to carbonic anhydrase. A formal Michaelis-Menten treatment of these enzyme-catalyzed hydrolyses shows that both k2 and Km vary with pH. The variation of Km with pH is shown to be dictated by the pH dependency of the turnover number (k2) while the apparent binding constant (k1/k-1) appears to be pH independent below 9. A comparison of the various esters reveals that binding increases with the size of the ester. The relative increase in free energy of binding associated with the larger esters is shown to be well accounted for by a parallel increase in the free energy of hydrophobic interactions. D2O studies indicate that k2 in D2O is twice as large as it is in H2O. © 1968, American Chemical Society. All rights reserved.