USE OF AROMATASE (CYP19) METABOLITE RATIOS TO CHARACTERIZE ELECTRON-TRANSFER FROM NADPH-CYTOCHROME-P450 REDUCTASE

Aromatase catalyzes the conversion of 4-androstene-3,17-dione to estro gen with the concomitant formation of the minor metabolites 4-androste ne-19-hydroxy-3,17-dione (19-hydroxyandrostenedione) and -androstene-3 ,17,19-trione(19-oxoandrostenedione). Microsomes of chinese hamster ov ary (CHO) cells expressing human aromatase were isolated to investigat e androstenedione metabolism. Relatively greater amounts of the minor metabolites result after limitation of electron flux from NADPH-cytoch rome P450 reductase to aromatase. Substitution of NADH for NADPH or li mitation of NADPH availability increased minor metabolite formation re lative to estrogen formation. Similar changes in metabolite ratios wer e observed when metabolism was conducted either at high pH (8.3) or in the presence of n-alcohols in the range of 5-200 mM alcohol concentra tions. However, conditions of low pH (5.5) or high ionic strength (1 M KCl) resulted in minor changes in metabolite ratios, suggesting littl e or no effect on electron flux between NADPH-cytochrome P450 reductas e and aromatase. Theoretical molar ratios of the resulting metabolites were predicted using a reaction scheme assuming sequential substrate oxidations without reversible intermediate release from the aromatase active site. This model was supported by a close agreement between the oretical and experimental metabolite ratios for a broad range of NADPH concentrations. The results indicate that metabolite ratios provide a sensitive indicator of aromatase-oxidoreductase interactions in the m icrosomal environment.