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Kinetic analysis of steps involved in oxidation by cytochrome P450 19A1
Author(s) -
Sohl Christal Dyane,
Guengerich F. Peter
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.512.7
Subject(s) - chemistry , aromatase , estrone , monooxygenase , steady state (chemistry) , aromatization , androstenedione , cytochrome p450 , catalysis , substrate (aquarium) , enzyme kinetics , stereochemistry , enzyme , biochemistry , active site , hormone , organic chemistry , medicine , androgen , biology , cancer , breast cancer , ecology
Cytochrome P450s are heme‐containing monooxygenases that catalyze the oxidation of endogenous compounds (e.g. vitamins and steroids) and exogenous compounds (e.g. drugs and pollutants). P450 19A1, or aromatase, is responsible for the conversion of androgens (androstenedione or testosterone) to estrogens (estrone or 17β‐estradiol). The reaction proceeds in three sequential steps, generating first a 19‐hydroxy‐ and then a 19‐aldo‐intermediate prior to the final aromatization step. Much study has focused on the inhibition of P450 19A1 because aromatase inhibitors have proven to be successful therapy for estrogen‐dependent breast cancer. However, few steady‐state studies on catalysis involve endogenous substrates and pre‐steady‐state kinetic characterization has not been done. We have developed a procedure for consistent heterologous bacterial expression and purification of P450 19A1. K d values for androstenedione, 19‐hydroxyandrostenedione, 19‐aldoandrostenedione, and estrone were measured to be in the very low μM range. Stopped‐flow spectroscopy indicated that binding of these ligands includes a fast spectroscopically silent (in absorbance) step, followed by a slower conformational change (E + L ⇔ EL ⇔ LE), which can be fit to double exponential curves. Single turnover (rapid chemical quench) yield progress curves that show temporal loss of substrate and formation of products and intermediates, generating rate constants for the sequential steps of catalysis. Global fitting of these steady state and pre‐steady state data allow the generation of the first kinetic models of the overall mechanism of P450 19A1 catalysis. Supported by USPHS R37 CA090426, T32 ES001590.