title: Chiral recognition at cytochrome P450 1A2 active site: effects of mutations at the putative distal site on the bindings of asymmetrical axial ligands.
abstract: Effects of mutations at the putative distal site of cytochrome P450 1A2 on chiral discrimination for binding (R)-(+)- and (S)-(-)-1-(1-naphthyl)ethylamine (ligand I), (R)-(-)- and (S)-(+)-1-cyclohexylethylamine (ligand II), and (R)-(+)- and (S)-(-)-1-(4-pyridyl)ethanol (ligand III) were studied by optical absorption spectra. The wild-type P450 1A2 exhibited different dissociation constants (Kd) for the R- and S-enantiomers of these ligands. The R/S ratios of the Kd values for ligands I and II were 5.2 and 2.9, respectively, and the S/R ratio for ligand III was 6.0. Mutations at the putative distal site, such as Glu318Asp and Glu318Ala, remarkably enhanced the discrimination: the R/S ratio of the Kd values for ligand I increased from 5.2 to 20-60, while the R/S ratio for ligand II decreased from 2.9 to 0.8-0.9. These remarkable changes in the R/S ratios were not observed with Glu318Asp mutation for ligand III binding, whereas affinities for both enantiomers of ligand III were markedly decreased by the Glu318Ala mutation. Mutation Thr319Ala increased the R/S ratio of the Kd values for ligand I slightly but markedly decreased the R/S ratio of ligand II (from 2.9 to 0.8) and the S/R ratio of ligand III (from 6.0 to 1.0). Similar enhancements of the chiral discriminations were observed with the mutation Lys250Leu at another putative substrate-recognition site. Differences between the R- and S-enantiomers of the standard enthalpy and entropy of ligand III binding were changed most remarkably by the Thr319Ser mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
authors.forename: A G
authors.initials: AG
authors.lastname: Krainev
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
authors.forename: M
authors.initials: M
authors.lastname: Ishigooka
authors.forename: K
authors.initials: K
authors.lastname: Hiroya
authors.forename: M
authors.initials: M
authors.lastname: Hatano
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005021:Ethylamines / Q000378:metabolism; D006736:Horses; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D008861:Microsomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D005021:Ethylamines; D008024:Ligands; D009281:Naphthalenes; D011725:Pyridines; D011994:Recombinant Proteins; C080150:1-(4-pyridyl)ethanol; C055450:1-(1-naphthyl)ethylamine; C080149:1-cyclohexylethylamine; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2