SITE-DIRECTED MUTAGENESIS OF THE PUTATIVE ACTIVE-SITE OF HUMAN 17-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-1

Several amino acid residues (Cys(54), Tyr(155), His(210), His(213) and His(221)) at a putative catalytic site of human 17 beta-hydroxysteroi d dehydrogenase type 1 were mutated to Ala. Replacement of His(221) by Ala remarkably reduced the catalytic activity, which resulted from a change of both the K-m and the V-max values of the enzyme. Compared wi th the wild-type enzyme, the catalytic efficiency of the His(221)-->Al a mutant was reduced 20-fold for the oxidative reaction and 11-fold fo r the reductive reaction. With similar mutations at His(210) Or His(21 3), no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residue s decreased the V-max. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys(54) ha s been localized in the cofactor-binding region of the enzyme, a Cys(5 4)-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr(155), which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Ty r(155)-->Ala, His(210)-->Ala + His(213)-->Ala and His(221)-->Ala mutat ions were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lowered catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or, remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala(237 )<->Val and Ser(312)<->Gly) were not found to affect either the cataly tic or the immunological properties of the type 1 enzyme.