Site-directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase type 1.
Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->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 Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower 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 (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.
Puranen
T J
TJ
Department of Clinical Chemistry, University of Oulu, Finland.
Poutanen
M H
MH
Peltoketo
H E
HE
Vihko
P T
PT
Vihko
R K
RK
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary; D018014:Gene Transfer Techniques; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015067:17-Hydroxysteroid Dehydrogenases; C032690:3-alpha-(17-beta)-hydroxysteroid dehydrogenase (NAD(+))