Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase).
Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).
Yamazaki
T
T
Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashihiroshima 739, Japan.
Ohno
T
T
Sakaki
T
T
Akiyoshi-Shibata
M
M
Yabusaki
Y
Y
Imai
T
T
Kominami
S
S
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004926:Escherichia coli; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008956:Models, Chemical; D011510:Proteolipids / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*
D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D013254:Steroid 17-alpha-Hydroxylase