Roles of phenylalanine at position 120 and glutamic acid at position 222 in the oxidation of chiral substrates by cytochrome P450 2D6.
The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1''-hydroxybufuralol with substrate enantioselectivity of (R)-BF >> (S)-BF and metabolite diastereoselectivity of (1''R)-OH < (1''S)-OH. The substitution of Phe-120 by alanine markedly increased the apparent K(m) and V(max) values for enantiomeric BTL 4-hydroxylation by CYP2D6. In contrast, the same substitution caused an increase only in V(max) values of (S)-BF 1''-hydroxylation without changing apparent K(m) values, while kinetic parameters (K(m) and V(max) values) for (R)-BF 1''-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling.
Masuda
Kazufumi
K
Laboratory of Pharmaceutical Physical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Tamagake
Keietsu
K
Katsu
Takashi
T
Torigoe
Fumihiro
F
Saito
Keita
K
Hanioka
Nobumitsu
N
Yamano
Shigeru
S
Yamamoto
Shigeo
S
Narimatsu
Shizuo
S
D000818:Animals; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry*; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
D018698:Glutamic Acid; D010649:Phenylalanine; D019389:Cytochrome P-450 CYP2D6