Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase.
The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.
Sawada
H
H
Department of Biochemistry, Gifu Pharmaceutical University.
Hara
A
A
Ohmura
M
M
Nakayama
T
T
Deyashiki
Y
Y
D015067:17-Hydroxysteroid Dehydrogenases; D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D006224:Cricetinae; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013237:Stereoisomerism
D009243:NAD; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; D015096:3-Hydroxysteroid Dehydrogenases; D006913:Hydroxysteroid Dehydrogenases; C032690:3-alpha-(17-beta)-hydroxysteroid dehydrogenase (NAD(+)); D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)