Sawada H, Hara A, Ohmura M, Nakayama T, Deyashiki Y
Department of Biochemistry, Gifu Pharmaceutical University.
J Biochem. 1991 May;109(5):770-5. doi: 10.1093/oxfordjournals.jbchem.a123455.
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.
对从仓鼠肝脏胞质溶胶中纯化得到的依赖NADP(+)的3α-羟基类固醇脱氢酶和依赖NAD(+)的3α(17β)-羟基类固醇脱氢酶的动力学机制进行了双向研究。对于3α-羟基类固醇脱氢酶，初始速度和产物抑制研究表明，酶反应顺序是有序的，NADP+与游离酶结合，NADPH是最后释放的产物。Cibacron蓝和己烯雌酚的抑制模式以及辅酶和底物的结合研究也与有序的双底物双产物机制一致。对于3α(17β)-羟基类固醇脱氢酶，稳态动力学测量和底物结合研究表明底物的结合模式是随机的，产物的释放是有序的；NADH最后释放。然而，这两种酶都将NAD(P)H的前R-氢原子转移到羰基底物上。
