Kinetic Studies of Cytochrome P-45017.alpha.,lyase Dependent Androstenedione Formation from Progesterone

Abstract

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-45017 alpha, lyase and NADPH-cytochrome P-450 reductase using a rapid quenching device at 10 degrees C. In these rapid quenching experiments, only the metabolites of [3H]progesterone bound to P-45017 alpha, lyase at the initial stage were detectable during the limited cycles of the P-45017 alpha, lyase reactions (1-120 s). The level of 17 alpha-hydroxy[3H]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [3H]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[3H]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-45017 alpha, lyase during the successive hydroxylation reaction into androstenedione. A kinetic model can clearly describe the successive reaction catalyzed by P-450 17 alpha, lyase, in which progesterone is converted successively into androstenedione via 17 alpha-hydroxyprogesterone, some of which dissociates from the active site of P-450 17 alpha, lyase and is never metabolized into androstenedione. We analyzed the effects of pH and the amount of NADPH-cytochrome P-450 reductase on the successive reaction and proved that the reaction was regulated by the rate of electron transfer for the conversion of the bound 17 alpha-hydroxyprogesterone to androstenedione. Furthermore, we found that the product dissociation from P-450 17 alpha, lyase is the rate-limiting process in the steady-state metabolism of progesterone by P-450 17 alpha, lyase.

Identification of Outer Mitochondrial Membrane Cytochrome b5 as a Modulator for Androgen Synthesis in Leydig Cells

“…These indicate that the first hydroxylation step is relatively fast and that the following steps (i.e. the release of the intermediate and C-C bond cleavage) are rate-limiting as shown previously (28). The increase of the electron transfer should enhance the second P-450-catalyzed reaction (i.e.…”

“…This system is believed to provide membrane proteins with a reaction field comparable with the biomembrane environment and allowed us to perform quantitative analyses in such a way as controlling the enzyme, modulator, and substrate concentrations. Androgen syntheses from progesterone by guinea pig P-450 17␣ (28) and from pregnenolone by bovine P-450 17␣ (29) are proven to proceed by a successive reaction in the presence of excess amounts of progesterone, in which efficient electron supplies from P-450 reductase to P-450 facilitate the C17-C20 cleavage reaction of the 17␣-hydroxylated intermediates bound to the enzyme pocket. The stimulatory effect of OMb on rat P-450 17␣ -catalyzed reactions was also observed (Fig.…”

“…Kinetic studies employing a rapid quenching method proved that androgen formation from progesterone by guinea pig P-450 17␣ and dehydroepiandrosterone formation from pregnenolone by bovine P-450 17␣ proceeded successively (28,29). In the successive reaction, rates of release of the intermediate from the substrate-binding pocket in the enzyme and the C-C bond cleavage together with release of the C17 ketosteroid determine yields of both the 17␣-hydroxyl and 17-ketosteroids.…”

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

“…Yamazaki et al (24,25) concluded that the reactions were processive in cultured bovine adrenocortical cells and rat ovarian microsomes. Tagashira et al (26) concluded that the oxidation of progesterone to androstenedione by guinea pig P450 17A1 was processive and that the rate-limiting step was product dissociation. Yamazaki et al (19), using similar approaches in the same laboratory, concluded that ϳ20% of 17␣-OH pregnenolone did not dissociate from bovine P450 17A1 in the oxidation of pregnenolone to DHEA.…”

“…Others have concluded that the enzyme is either distributive (20) or rather processive (19,(21)(22)(23)(24)(25)(26), with some of the results depending on the animal model.…”