Proximity of the Substrate Binding Site and the Heme-Iron Catalytic Site in Cytochrome P-450scc

As an approach to ``mapping'' the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1982-10, Vol.79 (19), p.5773-5777
Main Authors: Sheets, Joel J., Vickery, Larry E.
Format: Article
Language:English
Subjects:
Active sites
Adrenal Cortex - metabolism
Amides
Amines
Animals
Binding Sites
Cattle
Cholesterol Side-Chain Cleavage Enzyme - metabolism
Cholesterols
Cytochrome P-450 Enzyme System - metabolism
cytochrome P-450scc
Cytochromes
Enzymes
Heme - metabolism
Iron - metabolism
Kinetics
Ligands
Mitochondria - metabolism
Oxygen
Protein Binding
Spectrophotometry
Steroids
Titration
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Summary:As an approach to ``mapping'' the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23, 24-bisnor-5-cholen-3β -ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.79.19.5773