The aromatase active site: the C-6 "front" side of the androgen molecule is required for binding

Solubilized microsomes from human placenta were partially purified by anion-exchange chromatography on a quaternary methylamino-silica column. The aromatase-active fractions were then used for measuring optical difference spectra with various androgens, substituted at positions C-1, 2, 6, 11, 16, 17, and 19. Of these, only androgens substituted at the C-6, or frontside of the steroid backbone induce a strong type I spectrum, as well as competitive inhibition with the natural substrate, 4-androstene-3,17-dione. An in-house made affinity support, synthesized by reacting 6 beta-bromo-androstenedione with aminohexyl-Sepharose at pH 10-11 via a SN2-mechanism, was unable to trap the aromatase cytochrome P-450 component of the enzyme. A 3-dimensional model of the active site accounting for these interactions is proposed.