The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase.
An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.
J S
JS
Campbell
P J
PJ
Bertics
H J
HJ
Karavolas
D000818:Animals; D005260:Female; D007700:Kinetics; D008433:Mathematics; D008861:Microsomes / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D051381:Rats
D009249:NADP; D010088:Oxidoreductases; C022447:3-oxo-5-alpha-steroid 4-dehydrogenase (NADP(+))