Journal article
A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation
Archives of biochemistry and biophysics, Vol.416(1), pp.9-16
08/01/2003
Handle:
https://hdl.handle.net/2376/115935
PMID: 12859976
Abstract
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.
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Details
- Title
- A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation
- Creators
- Dan A Rock - Department of Chemistry, Washington State University, Pullman, WA 99164, USABrandon N S PerkinsJan WahlstromJeffrey P Jones
- Publication Details
- Archives of biochemistry and biophysics, Vol.416(1), pp.9-16
- Academic Unit
- Chemistry, Department of
- Publisher
- United States
- Grant note
- GM061823 / NIGMS NIH HHS ES 09122 / NIEHS NIH HHS
- Identifiers
- 99900547994901842
- Language
- English
- Resource Type
- Journal article