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 Michae...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 50mmol/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 kcat.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.展开更多
基金supported by grants from the Natural Sciences and Engineering Research Council(NSERC)Canada,and the Rhodes Trust(JY)the Higher Educational Funding Council for England.
文摘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 50mmol/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 kcat.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.
