Inhibition of Human Erythrocyte Carbonic Anhydrase B by Chloroacetyl Sulfonamides with Labeling of the Active Site

In an attempt to characterize the active site of human erythrocyte carbonic anhydrase B, we have studied the inhibition of this enzyme by several specific sulfonamide inhibitors and their N -chloroacetyl derivatives. In addition, two of the modified inhibitors were found to react irreversibly with the enzyme, resulting in inactivation. Enzymatic activity was determined from its catalytic hydrolysis of p -nitrophenyl acetate. The CO 2 -hydrating activity was also determined in some experiments and paralleled the activity determined from the esterase assays. N -Chloroacetylation at an amino group on the inhibitor far from the sulfonamide group resulted in little change or a decrease in the K i values of the two inhibitors tested. Modification at the sulfonamide group abolished the inhibitory ability of two inhibitors, but two others retained their ability to inhibit the enzyme although the K i values were higher. These are the only amide N -substituted sulfonamides that have been clearly shown to inhibit carbonic anhydrase reversibly. These two inhibitors, chloroacetylchlorothiazide and chloroacetylcyclothiazide, also reacted slowly with 1 eq of histidine per molecule of enzyme and caused complete, irreversible inactivation. After removal of the zinc atom from the enzyme, chloroacetylchlorothiazide failed to undergo this specific irreversible reaction. It is thought that the reactive histidine must be at or near the active site.