Studies of the active site of cytochrome P-450scc with a high-affinity spin-labeled inhibitor.
The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000037:antagonists & inhibitors*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003900:Desoxycorticosterone / Q000031:analogs & derivatives / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D013113:Spin Labels
D000345:Affinity Labels; D065607:Cytochrome P-450 Enzyme Inhibitors; D013113:Spin Labels; D003900:Desoxycorticosterone; D002784:Cholesterol; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme