Structural alteration of mouse P450coh by mutation of glycine-207 to proline: spin equilibrium, enzyme kinetics, and heat sensitivity.
Mouse cytochrome P450coh is a high-spin haem protein which specifically catalyses coumarin 7-hydroxylase activity. A mutation of Gly-207 to Pro shifts the P450coh completely to the low-spin form, indicating that the sixth axial position of the haem is hexaco-ordinated with a water molecule in the mutant G207P. Moreover, the G207P mutation increases the Km value for coumarin 7-hydroxylase activity 100-fold and the Kd value for coumarin binding 200-fold. Conversely, the mutation decreases the Ki and Kd values 10- and 20-fold respectively when testosterone, a larger molecule, is used as a substrate. The results, therefore, are consistent with an idea that the substrate pocket may be larger in the mutant G207P than in the wild-type cytochrome P-450. A Gly-207 to Ala mutation (G207A) of P450coh (G207A), on the other hand, affects neither the spectral nor the enzymic properties of P450coh. Pro-207, through cis/trans isomerization or formation of a kink, may confer on the G207P a structural alteration of its substrate-haem pocket. Our previous studies [Iwasaki, Juvonen, Lindberg and Negishi (1991) J. Biol. Chem. 266, 3380-3382; Juvonen, Iwasaki and Negishi (1991) J. Biol. Chem. 266, 16431-16435] show that the residue at position 209 in P450coh resides close to the sixth axial position of the haem, and the spin equilibrium of the cytochrome P-450 shifts toward the high-spin state as residue 209 becomes more hydrophobic and larger. A Gly-207 to Pro mutation, therefore, results in the creation of a larger substrate pocket in the mutant cytochrome P-450 by altering the protein structure around residue 209 so that a water molecule and testosterone can be accommodated.
Pharmacogenetics Section, National Institutes of Health, Research Triangle Park, NC 27709.
R O
RO
Juvonen
M
M
Iwasaki
T
T
Sueyoshi
M
M
Negishi
D000818:Animals; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004247:DNA; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005998:Glycine / Q000737:chemistry*; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D013379:Substrate Specificity
D007527:Isoenzymes; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D005998:Glycine