Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3.
The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.
Cowart
L A
LA
Department of Medicine, Vanderbilt University Medical, Nashville, Tennessee 37232, USA.
Falck
J R
JR
Capdevila
J H
JH
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D001426:Bacterial Proteins; D020134:Catalytic Domain / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007850:Lauric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D007527:Isoenzymes; D007850:Lauric Acids; C030358:lauric acid; C008757:palmitoleic acid; D016718:Arachidonic Acid; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D019255:NADPH Oxidases; D015126:8,11,14-Eicosatrienoic Acid