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| SITE-DIRECTED MUTAGENESIS OF THE PUTATIVE ACTIVE-SITE OF HUMAN 17-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-1 |
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| Author(s): PURANEN TJ, POUTANEN MH, PELTOKETO HE, VIHKO PT, VIHKO RK |
| Source: BIOCHEMICAL JOURNAL Volume: 304 Pages: 289-293 Part: Part 1 Published: NOV 15 1994 |
| Times Cited: 67 References: 38 |
| Abstract: Several amino acid residues (Cys(54), Tyr(155), His(210), His(213) and His(221)) at a putative catalytic site of human 17 beta-hydroxysteroid 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 with the wild-type enzyme, the catalytic efficiency of the His(221)-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His(210) Or His(213), no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the V-max. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys(54) has been localized in the cofactor-binding region of the enzyme, a Cys(54)-->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 Tyr(155)-->Ala, His(210)-->Ala + His(213)-->Ala and His(221)-->Ala mutations 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 catalytic or the immunological properties of the type 1 enzyme. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV OULU, BIOCTR, SF-90220 OULU, FINLAND
2. UNIV OULU, DEPT CLIN CHEM, SF-90220 OULU, FINLAND |
| Publisher: PORTLAND PRESS, 59 PORTLAND PLACE, LONDON, ENGLAND W1N 3AJ |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: PU039 |
| ISSN: 0264-6021 |
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