Enzyme kinetic and inhibition analyses of cytochrome P450XVII, a protein with a bifunctional catalytic site. Quantification of effective substrate concentrations at the active site and their significance for intrinsic control of the hydroxylase/lyase reaction sequence.
An enzyme kinetic analysis of the cytochrome P450XVII-dependent testicular steroid-17 alpha-hydroxylase/17,20-lyase system was performed which catalyzes simultaneously, at one active site, the formation of androstenedione from progesterone (productive pathway, coordinated performance of both hydroxylase and lyase activities) and the formation of 17 alpha-hydroxyprogesterone as the intermediate (abortive pathway, isolated performance of hydroxylase activity only). Progesterone was used as the substrate and promegestone (17 alpha, 21-dimethyl-4,9(10)-pregnadiene-3,20-dione) or estradiol as competitive inhibitors, and description of the data was based on the discrimination of those alternative routes of progesterone metabolism. The overall catalytic activity of P450XVII obeyed Michaelis-Menten kinetics (control incubations: V = 440 nM/min with 80 nM P450XVII, Km = 140 nM), and KI values amounted to 6.8 microM for promegestone and to 23.5 microM for estradiol. In contrast, analysis of the rates of both abortive and productive events yielded curvilinear Eadie-Hofstee plots. The former presented an apparent negative cooperative behavior with nh = 0.78 in the region of Kh, while the latter presented an apparent substrate inhibition behavior with the maximal rate occurring at that progesterone concentration where nh for the abortive path reached its minimum. Consequently, the ratio of productive versus abortive catalytic events decreased with increasing substrate supply, but did not reach zero. Effective substrate concentrations [S]eff with respect to either the productive or the abortive pathway were derived from Hill plots. Linearization of Eadie-Hofstee plots and parallel calculation methods using these [S]eff instead of [S] yielded V = 236 nM/min and [S]eff(0.5) = 131 nM for the abortive events and V = 217 nM/min and [S]eff(0.5) = 38 nM for the productive events. Both promegestone and estradiol were identified as competitive inhibitors of either reaction after consideration of effective progesterone concentrations. With the inhibitors, ratios of productive versus abortive events were constantly higher at a given substrate concentration than in control incubations, although neither promegestone nor estradiol affected progesterone or 17 alpha-hydroxyprogesterone accumulation in the endoplasmic reticulum membrane compartment. Rather, a linear correlation between ratios of productive versus abortive events and the overall catalytic rate as a measure of E.S complex concentration was obtained irrespective of the absence or presence of inhibitor. It was therefore concluded that promegestone and estradiol inhibit access or binding of progesterone to P450XVII, whereas the relative efficiency of androgen formation is solely dictated by the local substrate concentration being effective with respect to E.S formation. A model of P450XVII function is presented which proposes that excess substrate at the active site of the P450XVII enzyme protein hinders a certain fraction of a putative transient intermediate from being retained at the bifunctional catalytic site with the consequence that it cannot be further processed to androgen.
Kühn-Velten
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Institut für Physiologische Chemie, Universitätsklinikum, Essen, Federal Republic of Germany.
Bunse
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Förster
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D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011397:Promegestone / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013737:Testis / Q000201:enzymology
D003432:Cross-Linking Reagents; D065607:Cytochrome P-450 Enzyme Inhibitors; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D011397:Promegestone; D006899:Mixed Function Oxygenases; D008190:Lyases