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Engstrand, C, Jonsson, BH and Lindskog, S (1995) Catalytic and inhibitor-binding properties of some active-site mutants of human carbonic anhydrase I. Eur. J. Biochem. 229:696-702

Three isozyme-specific residues in the active site of human carbonic anhydrase I, Val62, 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 CO2 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 pKa 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.

PubMed

Amino Acids/chemistry; Binding Sites; Carbonic Anhydrase Inhibitors/metabolism; Carbonic Anhydrases/chemistry; Carbonic Anhydrases/genetics; Carbonic Anhydrases/metabolism; Catalysis; Humans; Isoenzymes/chemistry; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; Magnetic Resonance Spectroscopy; Mutagenesis, Site-Directed; Mutation; Structure-Activity Relationship