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

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Abstract

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.

Original languageEnglish
Pages (from-to)2998-3002
JournalJournal of Clinical Endocrinology and Metabolism
Volume94
Issue number8
Early online date26 May 2009
DOIs
Publication statusPublished - Aug 2009

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Aromatase
Enzyme Inhibitors
Genes
Kinetics
Enzyme activity
Substrates
Enzymes
Aminoglutethimide
letrozole
Cytochrome P-450 Enzyme System
Mutagenesis
Aromatase Inhibitors
Androstenedione
HEK293 Cells
Site-Directed Mutagenesis
Inhibitory Concentration 50
In Vitro Techniques
Exons
Prostatic Neoplasms
Binding Sites

Cite this

@article{3d93c3a86bba42ceaaef0e9a475743e6,
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 = "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.",
author = "Emily Payne and Evan Ingley and Ian Dick and Scott Wilson and Charlie Bond and Richard Prince",
year = "2009",
month = "8",
doi = "10.1210/jc.2008-2309",
language = "English",
volume = "94",
pages = "2998--3002",
journal = "Journal of Endocrinology & Metabolism",
issn = "0021-972X",
publisher = "ENDOCRINE SOC",
number = "8",

}

TY - JOUR

T1 - In Vitro Kinetic Properties of the Thr201Met Variant of Human Aromatase Gene CYP19A1 : Functional Responses to Substrate and Product Inhibition and Enzyme Inhibitors

AU - Payne, Emily

AU - Ingley, Evan

AU - Dick, Ian

AU - Wilson, Scott

AU - Bond, Charlie

AU - Prince, Richard

PY - 2009/8

Y1 - 2009/8

N2 - 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.

AB - 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.

U2 - 10.1210/jc.2008-2309

DO - 10.1210/jc.2008-2309

M3 - Article

VL - 94

SP - 2998

EP - 3002

JO - Journal of Endocrinology & Metabolism

JF - Journal of Endocrinology & Metabolism

SN - 0021-972X

IS - 8

ER -