The dextrorotatory enantiomer of aminoglutethimide is 38 times more potent than the levoenantiomer in inhibiting aromatization of testosterone by human placental microsomes. The spectral affinity constant for microsomal cytochrome P-450 is 36 times greater for the d-enantiomer. Enzymatic inhibition and affinity are highly correlated for each of the isomers as well as for the racemic mixture. Spectral analysis of the interactions of the inhibitors with the substrate supports the evidence for participation of cytochrome P-450 in aromatization.

Absorbance changes in the spectrum of cytochrome P-450 were related to the inhibition of [26-14C]cholesterol oxidation to [14C]isocaproate and pregnenolone in mitochondria from bovine corpus luteum produced by two types of ligands. Nitrogenous inhibitors, such as aminoglutethimide, elicit an absorption maximum at about 427 nm and a minimum at about 393 nm (type II), while steroidal inhibitors, such as (20R)-20-(p-tolyl)-5-pregnene-3beta,20-diol (20-tolyl-pregnenediol), cause difference spectra with maximum at about 420 nm and minimum at about 390 nm (reverse type I). The magnitude of spectral change and the amount of inhibition of pregnenolone synthesis by aminoglutethimide are closely correlated at concentrations ranging from 5 to 750 muM and by the model steroid, 20-tolyl-pregnenediol, at concentrations from 0.5 to 25 muM. The responses are concentration dependent and linear over the range of effective concentrations. The concentrations of inhibitors for the half-maximal inhibition of pregnenolone biosynthesis are identical with the concentrations producing half-maximal spectral changes within experimental error. Displacement of substrate from cytochrome P-450 and/or stabilization of the redox potential subsequent to to the ligation of heme iron is proposed as the specific mechanism of cholesterol side chain cleavage inhibition. Finding, together, the two procedures offer a sensitive, specific, and accurate means of screening inhibitors of the cholesterol side chain cleavage system.

Because aminoglutethimide is a potentially important drug in the treatment of certain maligancies as well as fertility control, its stereoisomers were studied for binding to corpus luteum mitochondrial cytochrome P-450 and inhibition of cholesterol side chain cleavage. The binding affinity, determined from induced spectral changes, is 2.6 times greater for the d- than for the l-isomer. In the enzyme assay, the d-isomer is 2.5 times more potent as an inhibitor of cholesterol side chain cleavage than is the l-isomer. The extent of inhibition and the change in the absorptivity of the P-450-inhibitor complex are linearly related for both chiral and racemic forms. Thus, the active center of the enzyme is stereoselective for the enantiomers of aminoglutethimide.

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