Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7α-hydroxysteroid dehydrogenase from Escherichia coli

被引:50
作者
Tanabe, T
Tanaka, N
Uchikawa, K
Kabashima, T
Ito, K
Nonaka, T
Mitsui, Y
Tsuru, M
Yoshimoto, T
机构
[1] Showa Univ, Sch Pharmaceut Sci, Shinagawa Ku, Tokyo 1428555, Japan
[2] Nagasaki Univ, Sch Pharmaceut Sci, Nagasaki 8528521, Japan
[3] Nagaoka Univ Technol, Dept Bioengn, Nagaoka, Niigata 9402137, Japan
[4] Nagasaki Univ, Informat Sci Ctr, Nagasaki 8528521, Japan
关键词
catalytic mechanism; site-directed mutagenesis; steroid dehydrogenase; X-ray analysis;
D O I
10.1093/oxfordjournals.jbchem.a022159
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Escherichia coli 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH; EC 1.1.1.159) has been the subject of our studies, including the cloning of its gene, and determination of the crystal structures of its binary and ternary complexes [J. Bacteriol. 173, 2173-2179 (1991); Biochemistry 35, 7715-7730 (1996)]. Through these studies, the Ser146, Tyr159, and Lys163 residues were found to be involved in its catalytic action. In order to clarify the roles of these residues, we constructed six single mutants of 7 alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y159H), 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 constants 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 exhibited 13.3% of the wild-type enzyme activity. No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detected on X-ray crystallography. On the other hand, the K163I mutant showed 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 conformational change was detected on X-ray crystallography. In the cases of the S146A and S146H mutants, the activities fairly decreased, with 20.3 and 35.6% of k(cat) of the wild-type, respectively. The data presented in this paper confirm that Tyr159 acts as a basic catalyst, that Lys163 binds to NAD(H) and lowers the pK(a) value of Tyr159, and that Ser146 stabilizes the substrate, reaction intermediate and product in catalysis.
引用
收藏
页码:634 / 641
页数:8
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