The molecular mechanisms of two common polymorphisms of drug oxidation-evidence for functional changes in cytochrome P-450 isozymes catalysing bufuralol and mephenytoin oxidation

Abstract

Using the stereospecific metabolism of (+)- and (-)-bufuralol and (+)- and (-)-metoprolol as model reactions, we have characterized the enzymic deficiency of the debrisoquine/sparteine-type polymorphism by comparing kinetic data of subjects in vivo with their microsomal activities in vitro and with reconstituted activities of cytochrome P-450 isozymes purified from human liver. The metabolism of bufuralol in liver microsomes of in vivo phenotyped 'poor metabolizers' of debrisoquine and/or sparteine is characterized by a marked increase in Km, a decrease in Vmax and a virtual loss of the stereoselectivity of the reaction. These parameters apparently allow the 'phenotyping' of microsomes in vitro. A structural model of the active site of a cytochrome P-450 for stereospecific metabolism of bufuralol and other polymorphically metabolized substrates was constructed. Two cytochrome P-450 isozymes, P-450 buf I and P-450 buf II, both with MW 50,000 Da, were purified from human liver on the basis of their ability to metabolize bufuralol to 1'-hydroxy-bufuralol. However, P-450 buf I metabolized bufuralol in a highly stereoselective fashion ((-)/(+) ratio 0.16) as compared to P-450 buf II (ratio 0.99) and had a markedly lower Km for bufuralol. Moreover, bufuralol 1'-hydroxylation by P-450 buf I was uniquely characterized by its extreme sensitivity to inhibition by quinidine. Antibodies against P-450 buf I and P-450 buf II inhibited bufuralol metabolism in microsomes and with the reconstituted enzymes. Immunochemical studies with these antibodies with microsomes and translations in vitro of RNA from livers of extensive and poor metabolizers showed no evidence for a decrease in the recognized protein or its mRNA. Because the antibodies do not discriminate between P-450 buf I and P-450 buf II, both a decreased content of P-450 buf I or its functional alteration could explain the polymorphic metabolism in microsomes. The genetically defective stereospecific metabolism of mephenytoin was determined in liver microsomes of extensive and poor metabolizers of mephenytoin phenotyped in vivo. Microsomes of poor metabolizers were characterized by an increased Km and a decreased Vmax for S-mephenytoin hydroxylation as compared to extensive metabolizers and a loss of stereospecificity for the hydroxylation of S-versus R-mephenytoin. A cytochrome P-450 with high activity for mephenytoin 4-hydroxylation was purified from human liver. Immunochemical studies with inhibitory antibodies against this isozyme suggest the presence in poor-metabolizer microsomes of a functionally altered enzyme.

Chromosomal assignment of human cytochrome P‐450 (debrisoquine/sparteine type) to chromosome 22.

“…Thus it can be concluded that the gene locus for PSO is located on chromosome 22 in close vicinity to P1. Several human (Guengerich et al, 1986;Meyer et al, 1986). But so far the molecular mechanism has not been definitely elucidated.…”

Differential foetal development of the O‐ and N‐demethylation of codeine and dextromethorphan in man.

“…The implications of the foetal lack of cytochrome P450 IID6 are unknown. In adults, the only known implication is in the oxidation of a limited number of drug substrates (Davies et al, 1981;Gonzalez et al, 1988;Meyer et al, 1986). In some cases the deficiency of the IID6 isoenzyme leads to accumulation of the drug.…”

Polymorphisms of the CYP1B1 Gene as Risk Factors for Human Renal Cell Cancer

“…Chemical procarcinogenic compounds require metabolic activation by oxidative enzymes such as CYP1B1 to be transformed into potentially carcinogenic forms (6,7). Several studies have shown that polymorphisms of CYP1B1 induce hyperactivation of proteins and increase the incidence of several cancers (8,9). The human CYP1B1 gene is located in the 2p21-22 region and consists of three exons (one of which is noncoding) and two introns (7,10).…”