title: In vitro kinetic properties of the Thr201Met variant of human aromatase gene CYP19A1: functional responses to substrate and product inhibition and enzyme inhibitors.
abstract: BACKGROUND
  The T(201)M 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 T(201)M variant would alter the structure of the enzyme and thus would also affect function compared to wild-type human aromatase.
  
  
  METHODS
  HEK293 cells were transiently transfected with CYP19A1 wild-type or T(201)M 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 T(201)M 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 (IC(50): 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 T(201)M increases enzyme activity and reduces the inhibitory effect of aminoglutethimide. These findings identify the T(201)M 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.
authors.affiliation: Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Perth, Western Australia 6009. ejpayne@cyllene.uwa.edu.au
authors.forename: E J
authors.initials: EJ
authors.lastname: Payne
authors.forename: E
authors.initials: E
authors.lastname: Ingley
authors.forename: I M
authors.initials: IM
authors.lastname: Dick
authors.forename: S G
authors.initials: SG
authors.lastname: Wilson
authors.forename: C S
authors.initials: CS
authors.lastname: Bond
authors.forename: R L
authors.initials: RL
authors.lastname: Prince
mesh_terms: D000616:Aminoglutethimide / Q000494:pharmacology; D000735:Androstenedione / Q000378:metabolism; D001141:Aromatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047072:Aromatase Inhibitors / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D047072:Aromatase Inhibitors; D000616:Aminoglutethimide; D000735:Androstenedione; D001141:Aromatase