Abstract
22-Ketocholesterol binds with high affinity to purified, phospholipid vesicle-reconstituted cytochrome P-450scc. Binding, quantitated using reversal of cholesterol-induced absorbance changes in the Soret region of the enzyme, indicates an affinity 3-5 times greater than that for the normal substrate cholesterol. The ketosteroid cannot be hydroxylated at position 22 and thus acts as a potent inhibitor of cholesterol side-chain cleavage. Steady-state kinetics demonstrate competitive inhibition by this steroid and provide a KI value several-fold lower than the cholesterol Km. On the basis of recently proposed mechanisms for hydroxylation by cytochromes P-450, 22-ketocholesterol may exert its inhibitory effect by acting as a tightly bound analogue that resembles the enzyme-bound cholesterol from which a hydrogen has been abstracted from position 22.
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