The kinetic mechanism of the hypothalamic progesterone 5 alpha-reductase.
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.
Campbell
J S
JS
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Karavolas
H J
HJ
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D005260:Female; D007031:Hypothalamus / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011375:Progesterone Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D011374:Progesterone; D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases; D011375:Progesterone Reductase