FATTY-ACID DISCRIMINATION AND OMEGA-HYDROXYLATION BY CYTOCHROME-P450 4A1 AND A CYTOCHROME P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN

FATTY-ACID DISCRIMINATION AND OMEGA-HYDROXYLATION BY CYTOCHROME-P450 4A1 AND A CYTOCHROME P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN

Alterman M A; Chaurasia C S; Lu P; Hardwick J P; Hanzlik R P

Archives of Biochemistry and Biophysics

1995

1995-07

The omega-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions, To investigate the chemical basis for these features of omega-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of omega-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein, Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1, With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover, The fusion protein omega-hydroxylated methyl laurate and lauryl alcohol about 1/10th as efficiently as lauric acid, but it did not hydroxylate lauramide, 10-Methoxydecanoic acid underwent O-demethylation (via omega-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the omega-position, In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid omega-hydroxylation by 43% at equimolar concentrations, omega-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation, Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca, 14 Angstrom) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus, (C) 1995 Academic Press, Inc.

Biochemistry & Molecular Biology; Biophysics; catalytic properties; cytochrome p450 4a1; escherichia-coli; expression; fusion protein; gene family; inhibition; lauric acid; mechanisms; omega-hydroxylation; purification

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