ROLES OF THE SER146, TYR159, AND LYS163 RESIDUES IN THE CATALYTIC ACTION OF 7-ALPHA-HYDROXYSTEROID DEHYDROGENASE FROM ESCHERICHIA-COLI

The Escherichia coli 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSD H; EC 1.1.1.159) has been the subject of our studies, including the cl oning of its gene, and determination of the crystal structures of its binary and ternary complexes [J. Bacteriol. 173, 2173-2179 (1991); Bio chemistry 35, 7715-7730 (1996)]. Through these studies, the Ser146, Ty r159, and Lys163 residues were found to be involved in its catalytic a ction. In order to clarify the roles of these residues, we constructed six single mutants of 7 alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y 159H), Lys163-Arg (K163R), Lys163-Ile (K163I), Ser146-Ala (S146A), and Ser146-His (S146H), by site-directed mutagenesis. These mutants were overexpressed in E. coli WSD, which is a 7 alpha-HSDH null strain, and the expressed enzymes were purified to homogeneity. The kinetic const ants of the mutant enzymes were determined, and the structures of the Y159F, Y159H, and K163R mutants were analyzed by X-ray crystallography . The Y159F mutant showed no activity, while the Y159H mutant exhibite d 13.3% of the wild-type enzyme activity. No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detect ed on X-ray crystallography. On the other hand, the K163I mutant showe d just 5.3% of the native enzyme activity, with a 8.5-fold higher K-d. However, the K163R mutant retained 64% activity, and no remarkable co nformational change was detected on X-ray crystallography. In the case s of the S146A and S146H mutants, the activities fairly decreased, wit h 20.3 and 35.6% of k(cat) of the wild-type, respectively. The data pr esented in this paper confirm that Tyr159 acts as a basic catalyst, th at Lys163 binds to NAD(H) and lowers the pK(a) value of Tyr159, and th at Ser146 stabilizes the substrate, reaction intermediate and product in catalysis.