Campbell J S, Karavolas H J
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
J Steroid Biochem. 1989 Feb;32(2):283-9. doi: 10.1016/0022-4731(89)90265-3.
The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5 alpha-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent Km (progesterone) = 95.4 +/- 4.5 nM; apparent Kia(NADPH) = 9.9 +/- 0.7 microM). The plot of 1/v vs 1/progesterone intersected on the ordinate which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent Kis = 4.3 +/- 1.3 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 31.9 +/- 1.4 microM and apparent Kii = 145.4 +/- 15.5 microM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.
通过初速度、产物抑制和终产物抑制研究确定，雌性大鼠下丘脑NADPH连接的孕酮5α-还原酶的动力学机制为平衡有序序列机制。对初速度数据的分析产生了相交的双倒数图，表明是一种序列机制（表观Km（孕酮）= 95.4±4.5 nM；表观Kia（NADPH）= 9.9±0.7 μM）。1/v对1/孕酮的图在纵坐标上相交，这与平衡有序机制一致。NADP与NADPH以及NADP与孕酮的产物抑制研究也支持底物的有序添加。NADP对NADPH是竞争性抑制剂（表观Kis = 4.3±1.3 μM），对孕酮是非竞争性抑制剂（表观Kis = 31.9±1.4 μM，表观Kii = 145.4±15.5 μM）。这些抑制模式表明NADPH在孕酮之前结合。综合起来，这些分析表明辅因子NADPH以快速平衡的方式与酶结合，并且优先于孕酮的结合。
