[Reconstruction and study of a multi-enzyme system by 11 beta-hydroxylase steroids].
The reconstitution of the steroid 11 beta-hydroxylase system based on the homogeneous proteins isolated from bovine adrenocortical mitochondria, cytochrome P-450 (P-450 (11 beta), 19-20.5 nmol of heme P-450 per 1 mg of protein), adrenodoxin (Adx) and adrenodoxin reductase (AR) was carried out. The reconstitution of the multienzyme system requires the presence of a non-ionic detergent due to the high hydrophobicity of P-450 (11 beta). Low concentrations of Tween 20 (below 0.015% or 115 microM) stimulate the reaction of steroid 11 beta-hydroxylation by improving the hemoprotein solubility. With a further increase in the detergent concentration, the reaction is inhibited due to the inactivation of the cytochrome and its impaired interaction with Adx. The electron transfer activity of adrenodoxin reductase and the dienzyme AR-Adx complex does not change within the Tween 20 concentration range of 0-0.4%. In solutions with the optimal concentration of Tween 20 (0.010-0.015%), the concentrations of AR and Adx providing for the half-maximum hydroxylation activity are 9 nM for AR and 280 nM for Adx. It was shown that in a reconstituted 11 beta-hydroxylase system, 75% of the reducing equivalents are involved in the formation of oxygen radicals, whereas 25%--in hydroxylation. 74% of the radical species are, in their turn, formed in the active site of the hemoprotein, while 26%--in the Fe2S2 center of adrenodoxin. The radical formation process predominates over the 11 beta-hydroxylation within a wide range of Adx/cytochrome ratios, i.e., 1.0-100. The hydroxylation substrate induces a 4-fold increase in the electron transfer rate by stimulating the enzymatic reduction of P-450 (11 beta), but only 35% of the additional reduced equivalents are consumed by the 11 beta-hydroxylation and 65%--by the oxygen radical formation.
D000311:Adrenal Glands / Q000201:enzymology; D000325:Adrenodoxin / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification / Q000378:metabolism; D006900:Hydroxylation; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D009097:Multienzyme Complexes; D000325:Adrenodoxin; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase
Rekonstruktsiia i izuchenie mul'tifermentnoĭ sistemy 11beta-gidroksilirovaniia steroidov.