Cytochrome P450: Role in Steroid Metabolism and Mechanism of Inhibition of Catalytic Activity.

Abstract

Cytochrome P-450 is a highly versatile biological catalyst that catalyzes the oxygenation of physiologically important substrates such as steroids as well as a large variety of foriegn compounds, including drugs. Although numerous forms of this enzyme have been purified in this and other laboratories, much remains to be learned about the mechanism of catalysis of the cytochrome and the manner in which it is affected by specific inhibitors. The present studies are concerned with an apparent isotope effect in progesterone 16$\\alpha$-hydroxylation by cytochrome P-450, the mechanism of aromatase inhibition by bromosteroids, and inhibition of the cytochrome by phencyclidine, an agent that has been used at an anesthetic but has become a drug of abuse. A new and improved method for measuring progesterone 16$\\alpha$-hydroxylation was developed, that involves the release of tritium to water. An apparent isotope effect of about 2 was found for this activity. This discovery reveals that hydrogen abstraction prior to oxygen insertion is one of the rate-limiting steps in the hydroxylation of progesterone at the 16$\\alpha$-position. Aromatase is the terminal enzyme for estrogen synthesis, and its inactivation would be of therapeutic importance. 6$\\beta$-Bromo and rostenedione, but not the 6$\\alpha$-epimer, was found to be a mechanism-based inactivator for the aromatase in both in vitro and in vivo systems. The 6$\\alpha$-epimer, on the other h and , is one of the most potent competitive inhibitors known. Phencyclidine (PCP) was shown to selectively inactivate, in a metabolism-dependent manner, P-450 isozyme 2, the major phenobarbital-inducible isozyme from rabbit liver microsomes. PCP did not inactivate P-450 forms 3a, 3b, 4, or 6. The phencyclidine iminium ion, a known oxidative metabolite of PCP, was shown to inactivate P-450 form 2 in a metabolism-dependent manner. The inactivation of this cytochrome by PCP leads to the loss of the Soret absorption and ferrous-carbonyl absorption, thus indicating the destruction of the prosthetic heme moiety. These studies have aided in the underst and ing of the mechanism of action of P-450 cytochromes, especially in regard to steroid and PCP metabolism. Such selective inactivators may prove to be of biomedical importance.