KINETIC-STUDIES OF CYTOCHROME P-450(17-ALPHA,LYASE) DEPENDENT ANDROSTENEDIONE FORMATION FROM PROGESTERONE

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-450(17 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 [H-3]progesterone bound to P-450(17 alpha,lyase) at the initial stage were detectable during the limited cycles of the P-450(17 alpha,lyase) reactions (1-120 s). The level of 17 alpha-hydroxy[H-3]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [H-3]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[H-3]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-450(17 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).