Roles of phenylalanine at position 120 and glutamic acid at position 222 in the oxidation of chiral substrates by cytochrome P450 2D6

✍ Scribed by Kazufumi Masuda; Keietsu Tamagake; Takashi Katsu; Fumihiro Torigoe; Keita Saito; Nobumitsu Hanioka; Shigeru Yamano; Shigeo Yamamoto; Shizuo Narimatsu

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 350 KB
• Volume: 18
• Category: Article
• ISSN: 0899-0042
• DOI: 10.1002/chir.20246

✦ Synopsis

Abstract

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. © 2006 Wiley‐Liss, Inc. Chirality