A site-directed mutagenesis study of human placental aromatase.
Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-450cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128----Gln, His-128----Ala, Cys-299----Ala, Glu-302----Leu, Asp-309----Asn, Asp-309----Ala, and Ser-312----Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] androstenedione (or [1 beta-3H]androstenedione), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19-hydroxyandrostenedione and 19-oxoandrostenedione by reverse phase high performance liquid chromatography using [7-3H]androstenedione as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis.
Zhou
D J
DJ
Division of Immunology, Beckman Research Institute of the City of Hope, Durate, California 91010.
Korzekwa
K R
KR
Poulos
T
T
Chen
S A
SA
D000616:Aminoglutethimide / Q000494:pharmacology; D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000378:metabolism; D047072:Aromatase Inhibitors; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology*; D011233:Precipitin Tests; D011247:Pregnancy; D014162:Transfection
D047072:Aromatase Inhibitors; D000616:Aminoglutethimide; D001141:Aromatase