NMR studies of recombinant cytochrome P450cam mutants.
In the active center of cytochrome P450cam, Thr-252 is one of the conserved amino acid residues in the cytochrome P450 superfamily and plays a key role in the hydroxylation of camphor. T252A mutant, in which Thr-252 is replaced by alanine, consumed O2 at a rate comparable to that of the wild-type enzyme, whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-typed enzyme and H2O2 is the main product in the hydroxylation reaction. H2O2 was also yielded by the valine mutant and the consumption rate of O2 was much lower than that for the wild-type enzyme (Imai et al (1989) Proc Natl Acad Sci USA 86, 7823-7827). On the basis of the 1H- and 15N-NMR spectra, it was revealed that the anionic nature of the axial thiolate and the heme-environmental structures were substantially affected in the absence of d-camphor by the amino acid substitution at 252 Thr. In T252A mutant, however, the binding of camphor reduced these conformational alterations in the heme vicinity, probably due to the formation of interactions between camphor and enzyme. On the other hand, T252V mutant still exhibited large reduction of the anionic nature of the axial ligand in the presence of d-camphor and structural changes around heme were also enhanced, since the affinity of the valine mutant to d-camphor was low. These results imply that the hydrophobic and/or steric effects of the valine residue at 252 interfere with interactions around heme and camphor binding sites, which corresponds to the larger functional defects for T252V mutant.
Wakasugi
K
K
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Ishimori
K
K
Morishima
I
I
D002164:Camphor; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D002164:Camphor; D006859:Hydrogen; D019475:Camphor 5-Monooxygenase