STUDIES ON THE ACTIVE SITES OF CYTOCHROMES P-450

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The cytochrome P-450s are a family of isozymes capable of oxidizing a wide variety of both endogenous and exogenous compounds. Two characteristics of these enzymes make it possible for a limited number of isozymes to metabolize a vast and varied array of exogenous chemical compounds. The first is the generally broad substrate and regio-specificity presumably due to relatively nonspecific substrate binding characteristics and multiple binding orientations. The second is a versatile active oxygenating species that is capable of oxidizing a variety of functional groups.These characteristics are being explored with the ultimate goal of predicting how changes in composition and structure of drugs will alter metabolic pathways. While most cytochrome P-450s have low substrate specificity, isozymes used for the metabolism of endogenous substance can be very specific. An example of a high specificity isozyme is aromatase, the enzyme responsible for the conversion of androgens to estrogens. This project describes our attempts at defining the binding influences responsible for certain drug metabolizing P-450 isozymes and the electronic and protein interactions responsible for the unusual mechanism of the third oxidation of aromatase. Methods used in the project include recombinant DNA techniques, determination of enzyme kinetics, and molecular modelling techniques. We have studied the metabolism of testosterone by clones, chimeras and single point mutants of P-450IIA1, IIA2 and P-450b using expressed P-450s provided by Dr. Frank Gonzales and his associates (LMC,NCI) . These studies have revealed that 1) modification of a few amino acid residues in critical positions can markedly affect not only the turnover number and pattern of metabolites, but also the enzyme stability, 2) regions of the polypeptide involved in binding are different for different families of isozymes and 3) the tertiary structure of P-450cam may be a valid model for mammalian P-450s.