The kinetic mechanism of the hypothalamic progesterone 5α-reductase

Abstract

The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5α-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 K m (progesterone) = 95.4 ± 4.5 nM; apparent K ia (NADPH) = 9.9 ± 0.7±M). The plot of l/v vs l/progesterone intersected on the ordinale 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 K is = 4.3 ± 1.3μM) and a noncompetitive inhibitor versus progesterone (apparent K is = 31.9 ± 1.4μM and apparent K ii = 145.4 ± 15.5μM). 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.