CATALYTIC VERSATILITY OF ERYTHROCYTE CARBONIC ANHYDRASE .6. KINETIC STUDIES OF NONCOMPETITIVE INHIBITION OF ENZYME-CATALYZED HYDROLYSIS OF P-NITROPHENYL ACETATE

The esterase activity of bovine carbonic anhydrase has been found to be reversibly inhibited by a large variety of monovalent anions as well as by sulfonamides, amino acids, alcohols, phenol, acetonitrile, and acetone. Anion, sulfonamide, and alcohol inhibitions are noncompetitive with respect to substrate; the former two decrease with pH while alcohol inhibition is not affected by pH. Chloride ions act at a site which behaves like an electrophilic center of pka ca. 7.5. The enzyme forms 1:1 complexes with these anions which are inactive with respect to esterase activity, but still retain their original capacity to bind p-nitrophenyl acetate. Likewise sulfonamides produce inactivation by the formation of 1:1 enzyme-inhibitor complexes. Anions such as CNO-, I-, and HCO3- were found to compete with each other as well as with acetazolamide for the same or for a nearby interacting site which is presumably situated at or near the chelated zinc ion present in the native enzyme and hence near or at a neighboring imidazolium ion. This anion binding site is different from the ester binding site. The sulfonamide binding site actually consists of at least two regions, one a hydrophobic region which attracts the aromatic portion of the sulfonamide, the other a zinc ion containing region which is required for the strong binding of the inhibitor. The dissociation constant of the acetazol-amide-enzyme complex rises dramatically at pH >10 as if dependent upon a group in the enzyme of pKa ca. 11. Anion inhibition was found to follow the order CN-> HS- > CNO-, SCN-, N3- > I-, ClO4- > HCO3-, HSO3- > NO3- > Br- > AcO- > Cl- > F-. The order observed with the very strong anionic inhibitors appears to parallel their association constants with zinc, while the order observed with the moderate and weak anionic inhibitors appears to parallel the Hofmeister lyotropic series. The data suggest that the binding site for noncompetitive inhibitors is sensitive to two closely situated electrophilic (cationic) groups, one of pKa ca. 7 and the other of pKa > 10. These findings are shown to parallel the behavior of the esteratic site of carbonic anhydrase. © 1968, American Chemical Society. All rights reserved.