Use of kinetic isotope effects to delineate the role of phenylalanine 87 in P450(BM-3).
The substrate oxidation rates of P450(BM-3) are unparalleled in the cytochrome P450 (CYP) superfamily of enzymes. Furthermore, the bacterial enzyme, originating from Bacillus megaterium, has been used repeatedly as a model to study the metabolism of mammalian P450s. A specific example is presented where studying P450(BM-3) substrate dynamics can define important enzyme-substrate characteristics, which may be useful in modeling omega-hydroxylation seen in mammalian P450s. In addition, if the reactive species responsible for metabolism can be controlled to produce specific products this enzyme could be a useful biocatalyst. Based on crystal structures and the fact that the P450(BM-3) F87A mutant produces a large isotope in contrast to the native enzyme, we propose that phenylalanine 87 is responsible for hindering substrate access to the active oxygen species for nonnative substrates. Using kinetic isotopes and two aromatic substrates, p-xylene and 4,4'-dimethylbiphenyl, the role phenylalanine 87 plays in active-site dynamics is characterized. The intrinsic KIE is 7.3 +/- 2 for wtP450(BM-3) metabolism of p-xylene. In addition, stoichiometry differences were measured with the native and mutant enzyme and 4,4'-dimethylbiphenyl. The results show a more highly coupled substrate/NADPH ratio in the mutant than in the wtP450(BM-3).
Rock
Dan A
DA
Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Boitano
Anthony E
AE
Wahlstrom
Jan L
JL
Rock
Denise A
DA
Jones
Jeffrey P
JP
D019943:Amino Acid Substitution; D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins; D001713:Biphenyl Compounds / Q000138:chemical synthesis / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000032:analysis / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010649:Phenylalanine / Q000737:chemistry / Q000502:physiology*; D013379:Substrate Specificity / Q000235:genetics; D014992:Xylenes / Q000138:chemical synthesis / Q000378:metabolism
D001426:Bacterial Proteins; D001713:Biphenyl Compounds; D014992:Xylenes; D010649:Phenylalanine; D009249:NADP; C031286:4-xylene; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases