Inhibition and Chemical Modifications of Human Erythrocyte Carbonic Anhydrase B

Specific chemical modification studies were carried out in an attempt to characterize the "active site" of carbonic anhydrase with regard to its catalytic properties, the binding of inhibitors (anions and sulfonamides), and the binding and role of the zinc atom. Enzymatic activity was determined by catalytic hydrolysis of p -nitrophenyl acetate. The CO 2 -hydrating activity was also determined in some experiments, and it paralleled the activity determined from the esterase assays. Amidination of all the lysyl residues of the enzyme with methyl acetimidate hydrochloride caused no loss of activity. Hence, a lysine residue is not involved in any critical function at the active site. Bromoacetate and iodoacetamide both inactivate the enzyme by reaction at the 3'-imidazole nitrogen of a histidine residue. By measuring enzymatic activity immediately after addition of these reagents, both were found to be reversible inhibitors of enzymatic activity. Furthermore, the rate of inactivation is directly proportional to the fraction of the enzyme which is reversibly inhibited by these reagents. Other inhibitors, chloride and sulfanilamide, compete with the binding of iodoacetamide and competitively protect the enzyme against inactivation. The histidine (or histidines) must be located near the inhibitor-binding site and react with these reagents while they are bound there. The zinc atom may be removed from the native enzyme and replaced by cobalt to regenerate an active enzyme (Co(II)-enzyme). Inactivation of Co(II)-enzyme is similar to that of the native enzyme. The rate of inactivation of the apoenzyme (as measured in terms of activity after adding back zinc or cobalt) by iodoacetamide is about the same as with native or Co(II)-enzyme, but the rate with bromoacetate is only 1 to 2% of its rate with native or Co(II)-enzyme. In addition, chloride does not protect the apoenzyme against inactivation by iodoacetamide. Hence, the metal atom seems to be involved in the binding of anionic inhibitors. Inactivation by these reagents is incomplete, and evidence is presented which suggests that the residual activity is a function of the modified enzyme. The residual activity after reaction of the native enzyme with iodoacetamide is about 16% of that of the native enzyme at pH 7.4. After reaction with bromoacetate, it amounts to about 4% at pH 7.5 and 15% at pH 9.6. Chloroacetamide also acts as a reversible inhibitor of carbonic anhydrase, but it does not inactivate the enzyme. 2,