Abstract
Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k(cat) of 0.06 s(-1) for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone.
Highlights
An unusual feature of some P450s is the ability to catalyze sequential reactions
There is agreement that catalysis occurs at one active site and must proceed through the 19-OH andro and 19ϭO andro intermediates [18], it remains unclear whether these intermediates dissociate over the course of the reaction
We found that one successful method of stabilizing P450 19A1 during purification was the addition of the 19A1 inhibitor ␣NF [44] during the solubilization and chromatography steps [23], an approach used previously with P450s 1A1 and 1A2 [32, 45]
Summary
Chemicals—Andro, 19-OH andro, 19ϭO andro, and estrone were purchased from Steraloids (Newport, RI), and all radiolabeled steroids were purchased from PerkinElmer (Waltham, MA). By centrifugation at 5000 ϫ g (20 min), the resulting spheroplast pellet was resuspended in Buffer B (100 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol (v/v), 6 mM magnesium acetate, and 0.1 mM dithiothreitol) To this were added the protease inhibitors leupeptin (2 M), aprotinin (0.04 unit/ ml), bestatin (1 M), and phenylmethylsulfonyl fluoride (1 mM final, from a 100 mM stock solution in 1-propanol). Measurement of Enzyme Activity—In general steady-state studies, the reconstituted enzyme system contained 0.10 M P450 19A1, 0.20 M NADPH-P450 reductase, and 9 M L-␣1,2-dilauroyl-sn-glycero-3-phosphocholine (dispersed into vesicles by sonication prior to use, as a 1 mg/ml stock solution), followed by the addition of substrate dissolved in CH3OH (final organic solvent content Յ 1% v/v). A reconstituted enzyme system containing 20 nM P450 19A1, 600 nM NADPH-P450 reductase, and 28 M L-␣-1,2-dilauroyl-sn-glycero-3-phosphocholine (dispersed into vesicles by sonication prior to use, as a 1 mg/ml stock solution) was incubated with 4.9 M [1-3H]andro (2 Ci) for 5 min (1.0-ml volume). HPLC (20-l injections) was used to separate the compounds using an octadecylsilane (C18) column (6.2 mm ϫ 80 mm, 3 m, Agilent Technologies, Palo Alto, CA)
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