A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation.
A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis-Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme-substrate (ES) complex responsible for omega hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to omega-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of omega to omega-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3 is able to produce the omega alcohol, a product that arises from the high-energy primary radical.
Rock
Dan A
DA
Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Perkins
Brandon N S
BN
Wahlstrom
Jan
J
Jones
Jeffrey P
JP
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D001671:Biochemistry / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D004734:Energy Metabolism; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity / Q000502:physiology
D001426:Bacterial Proteins; D007850:Lauric Acids; C030358:lauric acid; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases