Chiral recognition at cytochrome P450 1A2 active site: effects of mutations at the putative distal site on the bindings of asymmetrical axial ligands.

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)