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)

The role of Thr268 in oxygen activation of cytochrome P450BM-3

“…The role of threonine in microsomal P450d was suggested to be in the recognition of the substrate rather than the stabilization of the oxygen-bound complex. This is further supported from the studies using the asymmetrical axial ligand (Krainev et al, 1993) since Thr319 appears to be important for discriminating between chiral axial ligands.…”

Purification and characterization of rat liver enzyme catalyzing stereoselective reduction of acetylpyridines

“…13 3-Acetylpyridine (2) is known as a neurotoxin 14 and possesses niacin-like activity. 15 1-(4-Pyridyl)ethanol (4), the reduced form of 1, is recognized by nitric oxide synthase 16 and CYP 1A2 17 in a chiral discriminative manner. 1-(3-Pyridyl)ethanol (5), the reduced form of 2, is involved in the turnover of 7-14 C-nicotinamide dinucleotides in mice.…”

Activation of MeIQ (2-amino-3,4-dimethylimidazo- [4,5-f]quinoline) by sequence variants of recombinant human cytochrome P450 1A2

“…(1) The ionic region is defined by rat residues 250 KRFK 253 [Krainev et al, 1992]; the corresponding human P450 1A2 residues are 251 QRFK 254. (2) The distal region (rat P450 1A2 residues 310 -325) is believed to correspond to the long I-helix motif of P450 cam , forming part of the hydrophobic pocket surrounding the heme ring, on the opposite face to the cysteine thiolate ligand [Krainev et al, 1993]. (The ionic and distal regions correspond roughly to SRS3 and SRS4, respectively.)…”