Kinetic studies of cytochrome P-45017 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-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.
Tagashira
H
H
Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, Japan.
Kominami
S
S
Takemori
S
S
D019326:17-alpha-Hydroxyprogesterone; D000311:Adrenal Glands / Q000201:enzymology; D000446:Aldehyde-Lyases / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006908:Hydroxyprogesterones / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase
D006908:Hydroxyprogesterones; D000735:Androstenedione; D011374:Progesterone; D009249:NADP; D019326:17-alpha-Hydroxyprogesterone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D000446:Aldehyde-Lyases