title: Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system.
abstract: Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19 and a subsequent C19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Km values in the detergent and/or lipid environment compared with buffer alone. Although the apparent Km and Vmax values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Km was lower and the Vmax was higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.
authors.affiliation: Department of Biological Sciences, State University of New York, Buffalo 14260.
authors.forename: K
authors.initials: K
authors.lastname: Sethumadhavan
authors.forename: F L
authors.initials: FL
authors.lastname: Bellino
mesh_terms: D000728:Androgens / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D001141:Aromatase / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
chemical_list: D000728:Androgens; D013739:Testosterone; D000735:Androstenedione; D003577:Cytochrome P-450 Enzyme System; D001141:Aromatase; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen