Catalytic efficiency of expressed aromatase following site-directed mutagenesis.
Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate. Estimations of the actual turnover rate (catalytic efficiency) were derived 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 the Vmax observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the structure-function relationship of aromatase cytochrome P-450, the observed reduction of enzyme activity in terms of Vmax following site-directed mutagenesis led to the assumption that there was a corresponding loss of catalytic effectiveness. The present study reveals that a lower 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 content was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of the heme-binding property. Thus, interpretation of losses and reductions in aromatase activity resulting from single amino-acid replacement should take into account changes in the specific content of aromatase cytochrome P-450.
Kadohama
N
N
Medical Foundation of Buffalo Research Institute, NY 14203.
Zhou
D
D
Chen
S
S
Osawa
Y
Y
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004967:Estrogens / Q000096:biosynthesis; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D004967:Estrogens; D000735:Androstenedione; D006418:Heme; D001141:Aromatase