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

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. Loss-of-function mutations in aromatase (CYP19A1) have been characterized previously. A new Thr201Met variant, previously associated with reduced prostate cancer progression, demonstrates substantial gain of function in enzyme activity.