Structure-function studies of human aromatase by site-directed mutagenesis: Kinetic properties of mutants Pro-308 → Phe, Tyr-361 → Phe, Tyr-361 → Leu, and Phe-406 → Arg

Abstract

Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [1β,2β-3H]androstenedione, [19-14C]androstenedione, and [1β,2β-3H]testosterone as substrates. The mutant Phe-406 → Arg was completely inactive. Since there were only small changes in the Km and Vmax values for all substrates for mutants Tyr-361 → Phe and Tyr-361 → Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308 → Phe had altered catalytic properties; the Km values for androstenedione, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethunide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.