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 a nhydrase I, Val62, His67, and His200, have been changed by site-direct ed mutagenesis to their counterparts in human carbonic anhydrase II, A sn62, Asn67, and Thr200. A double mutant, containing Asn62 and Asn67, and a triple mutant, containing all three alterations, were also produ ced. The rates of CO2 hydration and ester hydrolysis catalyzed by thes e mutants, the inhibition of these enzymes by the anions, SCN-, and I- , and the binding of the sulfonamide inhibitors, dansylamide and MK-41 7 (a thienothiopyran-2-sulfonamide) have been measured. The results su ggest that the effect of His200 in isozyme I is to prolong the lifetim e of the enzyme-bicarbonate complex and to increase the pK(a) of the c atalytic group, a zinc-coordinated water molecule. For isozyme I, Val6 2 and His67 might interfere with the function of a proton 'shuttle' gr oup in the active site, thus maintaining the buffer specificity of a c ompulsory proton-transfer step. The single mutations have small effect s on anion binding. Only the triple mutant has anion-binding propertie s resembling those of isozyme II. All mutants show altered sulfonamide -binding properties. In particular, the binding specificity is affecte d. 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.