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In Vitro Kinetic Properties of the Thr²⁰¹Met Variant of Human Aromatase Gene CYP19A1: Functional Responses to Substrate and Product Inhibition and Enzyme Inhibitors

Department of Endocrinology and Diabetes (E.J.P., S.G.W., R.L.P.), Sir Charles Gairdner Hospital, Nedlands, Western Australia, 6009 Perth, Australia; and School of Medicine and Pharmacology (E.J.P., I.M.D., S.G.W., R.L.P.), Cell Signalling Group (E.I.), Laboratory for Cancer Medicine, Western Australia Institute for Medical Research and Centre for Medical Research, and School of Biomedical, Biomolecular, and Chemical Sciences (C.S.B.), University of Western Australia, Crawley, Western Australia, 6009 Perth, Australia

Address all correspondence and requests for reprints to: Emily Payne, Department of Endocrinology and Diabetes, First Floor, C Block, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Perth, Western Australia 6009. E-mail: ejpayne{at}cyllene.uwa.edu.au.

Context: The T²⁰¹M variant (rs28757184) within exon 5 of the human aromatase gene CYP19A1, present in up to 20% of some populations, has been reported to reduce prostate cancer progression.

Objective: We hypothesized that the T²⁰¹M variant would alter the structure of the enzyme and thus would also affect function compared to wild-type human aromatase.

Design: HEK293 cells were transiently transfected with CYP19A1 wild-type or T²⁰¹M variant gene transcripts made by site-directed mutagenesis and enzyme activity measured using tritiated androstenedione as the substrate. The effects of differing concentrations of substrate and product (E1 and E2) and four aromatase inhibitors were assessed.

Results: At all substrate concentrations tested, the T²⁰¹M variant showed substantially increased activity compared to the wild-type (Vmax: variant, 738 ± 36 pmol/h · mg; wild-type, 189 ± 17 pmol/h · mg, P < 0.0001; Km: variant, 64.4 ± 19.3 nM; wild-type, 46.6 ± 9.1 nM, P = 0.04). Kinetic analysis showed evidence of substrate inhibition for the wild-type, but no product inhibition was demonstrated for either transcript. Formestane, chrysin, and letrozole had no differential inhibitory effect on the two transcripts, but aminoglutethimide inhibition was substantially reduced in the variant compared to wild-type (IC₅₀: wild-type, 1.3 ± 0.2 nM; variant, 45 ± 14.2 nM, P = 0.002; and Ki: wild-type, 0.7 ± 0.2 nM; variant, 29.6 ± 9.7 nM, P = 0.0001).

Conclusions: In addition to loss of function mutations previously described, a new naturally occurring relatively common alteration of enzyme structure at T²⁰¹M increases enzyme activity and reduces the inhibitory effect of aminoglutethimide. These findings identify the T²⁰¹M site, distant from the substrate-binding site and not previously considered to play a role in enzyme activity, as a functionally important area of the enzyme that may play a role in the propensity to disease. Common to other cytochrome P450 enzymes, wild-type aromatase demonstrates substrate but not product inhibition.