title: Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1).
abstract: Fatty acid binding and oxidation kinetics for wild type P450(BM3) (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k (cat). The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
authors: Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK.
authors: Benjamin
authors: B
authors: Rowlatt
authors: Jake A
authors: JA
authors: Yorke
authors: Anthony J
authors: AJ
authors: Strong
authors: Christopher J C
authors: CJ
authors: Whitehouse
authors: Stephen G
authors: SG
authors: Bell
authors: Luet-Lok
authors: LL
authors: Wong
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D007850:Lauric Acids; D019814:Myristic Acid; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases