Abstract
Upon irradiation by a light flash (100-J), the carbon monoxide complex of cytochrome P-450scc was fully photodissociated in both the presence and absence of cholesterol, while less than 20% of the CO complex was photodissociable with those of deoxycorticosterone-bound and -free forms of cytochrome P-45011 beta. When the quantum yield of the reaction was measured for each photodissociable portion, the values were 0.5 and 1.0 for the substrate-free and -bound forms of cytochrome P-450scc, and 0.03 and 0.8 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. Thus, CO complexes of these enzymes become more photosensitive upon binding with the specific substrates. Steroid binding also affected kinetic constants of reactions between the ferrous enzymes and CO. The rate constants for the CO recombination at 15 degrees C were 2.7 X 10(6) and 2.3 X 10(5) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-450scc, and were 7.0 X 10(5) and 5.4 X 10(3) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. The rate constants for the CO dissociation also decreased upon the steroid bindings. The products of the enzyme reactions, pregnenolone and corticosterone, had similar effects on the kinetic constants. From these findings, we postulate that the binding of a steroid to the substrate site of each enzyme alters the bonding character of CO with the heme-iron, thereby affecting both photochemical and kinetic properties of the CO complex. The nature of the photoindissociable portion of the CO complex of cytochrome P-45011 beta is also discussed.
Highlights
Upon irradiation by a light flash(100-J),the carbon ases which catalyze the side-chain cleavage of cholesterol and monoxide complex of cytochrome P-45Oscc was fully the lip-hydroxylation of both deoxycorticosterone and 11
The two enzymes, together with cholesterol, while less than 20% of the CO complex was the members of a specific electron transport system for them photodissociable with those of deoxycorticosteronebound and -freeforms of cytochrome P-450118
CO complexes of these enzymes be- In thereactions catalyzed by these cytochromes P-450, the come more photosensitive upon binding with the spe- substrate to be monooxygenated serves as an effector of cific substrates
Summary
5-en-38-01-2O-one;deoxycorticosterone, 21-hydroxy-pregn-4-en-3,20dione; 11-deoxycortisol, 17a,21-dihydroxy-pregn-4-en-3,2O-dione; search Development Corporation of Japan, %,Osaka Medical College, corticosterone, 4-pregnen-11@,21-diol-3,20-dione1;8-hydroxydeoxy-. The purified preparations of P-45Oscc and P-450118 obtained as above contained a roughly stoichiometric amount of cholesterol and an excess amount of deoxycorticosterone, respectively; the former was that endogenously bound to P-45Oscc, and the latter was that added exogenously to stabilize P-45011p during the purification These steroidswere depleted from the enzyme preparations by incubating them with the followingreaction systems [19].For the depletion of cholesterol, a purified P-45Oscc preparation containing 20 nmol of the cytochrome was incubated with 0.5 nmol of adrenodoxin reductase, 5 nmol of adrenodoxin, 100 nmol of NADPH, 1pmol of glucose 6-phosphate, 0.7 unit of glucose-6-phosphate dehydrogenase in 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4, which contained 0.1 mM dithiothreitol, 0.1 mM EDTA, and 0.04% bovine serum albumin, at room temperature for 30 min under normal atmospheric conditions.
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