Covalent labeling of the active site of human carbonic anhydrase B with N-bromoacetylacetazolamide.

Two or possibly three equivalents of N-bromoacetylacetazolamide interact with two histidine residues located in the active site region of human carbonic anhydrase B. The most rapid reaction occurs at the l-nitrogen of a histidine residue and this is followed by a slower alkylation of the 3-nitrogen of the same histidine and the 3-nitrogen of a second histidine. From a reaction mixture of N-bromo[14C]acetylacetazolamide and the enzyme, a species was isolated that contained approximately 1 eq of reagent covalently bound to the lnitrogen of a histidine (or histidines). This alkylated enzyme is active toward p-nitrophenyl acetate, but shows a pH dependence which is different from that of the native enzyme. At pH 6 the esterase activity is 9.5%, and levels off at pH 7.6 to about 35% relative to the native enzyme. The K,, of the alkylated enzyme is almost identical with that of the native enzyme. These observations indicate that the histidine that reacted fastest with the reagent does not participate in the catalysis. Like the native enzyme alkylated human carbonic anhydrase B reacts with bromoacetate at the j-nitrogen position of a histidine in the active site region. The partially purified dialkylated enzyme has a residual esterase activity at pH 7.6 of less than 0.8%. This raises the possibility that the histidine that reacts with bromoacetate plays a role in the catalysis.