Functional evaluation of cytochrome P450 2D6 with Gly(42)Arg substitution expressed in Saccharomyces cerevisiae

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared wit h those of other single (P34S, R296C and S486T) and double amino acid-subst ituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cell s, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsom al fractions and their oxidation capacities towards debrisoquine as a proto typic substrate and bunitrolol as a chiral substrate were different from th ose of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved sim ilarly to the wild-type in these indices. The CYP contents both in yeast mi crosomal and in whole cell fractions indicated that some part of G42R prote in was localized in the endoplasmic reticulum membrane fraction, whereas mo st of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent K -m and decreased V-max for debrisoquine 4-hydroxylation, while it increased both K-m and V-max for bunitrolol 4-hydroxylation. The P34S substitution d id not drastically change K-m. but decreased V-max for debrisoquine 4-hydro xylation, whereas K-m was increased and V-max unchanged or decreased for bu nitrolol 4-hydroxylation by P34S substitution. These results suggest that t he G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution. Pharmacogen etics 11:709-718 (C) 2001 Lippincott Williams & Wilkins.