In short-term experiments, 6β-bromotestosterone acetate is a competitive inhibitor of the crystalline Δ5-3-ketosteroid isomerase (EC 126.96.36.199) of Pseudomonas testosteroni. Upon prolonged incubation, 6β-bromotestosterone acetate causes a time- and concentration-dependent irreversible inactivation of the enzyme which is favored at elevated pH values. Protection against this inactivation is afforded by the simultaneous presence of 19-nortestosterone, a truly competitive inhibitor of the enzyme. Upon incubation with radioactive 6β-bromotestosterone 17-[3H]acetate, the enzyme becomes labeled, and under suitable conditions more than one mole of steroid is bound to each mole of enzyme. These findings suggest that 6β-bromotestosterone 17-acetate is an active-site-directed irreversible inhibitor of Δ5-3-ketosteroid isomerase. At least three species of inactive enzyme separable by polyacrylamide gel electrophoresis are formed during the inactivation process. It seems likely that these species reflect the binding of progressively increasing numbers of steroid molecules, and that eventually the labeled enzyme dissociates into its subunits.
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