CATALYTIC EFFICIENCY OF EXPRESSED AROMATASE FOLLOWING SITE-DIRECTED MUTAGENESIS

Mutant aromatase cytochrome P-450s, expressed in CHO cells after trans fection with cDNAs, have been characterized in terms of their catalyti c efficiencies. After solubilization from microsomes, specific aromata se P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309 Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunoso rbent assay (ELISA). Microsomal aromatase activity was determined by t he H-3-water method using [1beta-H-3]androstenedione as substrate. Est imations of the actual turnover rate (catalytic efficiency) were deriv ed from the combined data. The P-450 content in the mutants varied but was always less than that in the wild type. Hence, the decreases in t he V(max) observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the s tructure-function relationship of aromatase cytochrome P-450, the obse rved reduction of enzyme activity in terms of V(max) following site-di rected mutagenesis led to the assumption that there was a correspondin g loss of catalytic effectiveness. The present study reveals that a lo wer P-450 content can contribute significantly to decreasing catalytic activity in the mutants. In fact, in mutant Phe406Arg which exhibited virtually no catalytically active aromatase, the specific P-450 conte nt was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of t he heme-binding property. Thus, interpretation of losses and reduction s in aromatase activity resulting from single amino-acid replacement s hould take into account changes in the specific content of aromatase c ytochrome P-450.