Catalytic and Inhibitor‐Binding Properties of Some Active‐Site Mutants of Human Carbonic Anhydrase I

Three isozyme‐specific residues in the active site of human carbonic anhydrase I, Va162, His67, and His200, have been changed by site‐directed mutagenesis to their counterparts in human carbonic anhydrase II, Asn62, Asn67, and Thr200. A double mutant, containing Asn62 and Asn67, and a triple mutant, containing all three alterations, were also produced. The rates of CO 2 hydration and ester hydrolysis catalyzed by these mutants, the inhibition of these enzymes by the anions, SCN − , and I − , and the binding of the sulfonamide inhibitors, dansylamide and MK‐417 (a thienothiopyran‐2‐sulfonamide) have been measured. The results suggest that the effect of His200 in isozyme I is to prolong the lifetime of the enzyme‐bicarbonate complex and to increase the p K a , of the catalytic group, a zinc‐coordinated water molecule. For isozyme I, Val62 and His67 might interfere with the function of a proton ‘shuttle’ group in the active site, thus maintaining the buffer specificity of a compulsory proton‐transfer step. The single mutations have small effects on anion binding. Only the triple mutant has anion‐binding properties resembling those of isozyme II. All mutants show altered sulfonamide‐binding properties. In particular, the binding specificity is affected. While wild‐type isozyme I binds dansylamide 50 times more strongly than MK‐417, the triple mutant shows a reversed selectivity and binds MK‐417 nearly 50 times more strongly than dansylamide.