Studies of the esterase activity and the anion inhibition of bovine zinc and cobalt carbonic anhydrases.

Abstract

The rates of hydrolysis of nitrophenyl esters, catalyzed by bovine carbonic anhydrase, depend on the position of the nitro group and on the size of the acyl residue. The most rapidly hydrolyzed substrate of those investigated is p‐nitrophenyl acetate. The catalyzed rates are proportional to both enzyme and ester concentrations. Only in the case of m‐nitrophenyl acetate could a value of the Michaelis constant, Km, be estimated, approximately 10 mM. Product inhibition by o‐nitrophenol occurs during the enzyme‐catalyzed hydrolysis of o‐nitrophenyl acetate. The metal specificity of the enzyme seems to be independent of the substrate. Zn(II) and Co(II) are about equally effective in restoring esterase activity to the apoenzyme while all other metal ions studied do not activate or have a very small effect. The pH‐dependence of the esterase activity of both Zn(II)‐ and Co(II)‐carbonic anhydrase is very similar to that of the CO2 hydration activity, and the basic form of a group with a pK near 7 is required for both reactions. Anionic inhibitors are strongly bound to the enzyme when this group is in its acidic form, but the inhibition is almost abolished at alkaline pH. The inhibitory powers of cyanide and sulfide decrease at acid as well as at alkaline pH. The results agree with the assumption that, formally, CN− and HS− do not bind to the basic form of the enzyme, while HCN and H2S do not bind to the acidic form.