Functional evaluation of cytochrome P450 2D6 with Gly42Arg 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 with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, 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 microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.
Tsuzuki
D
D
Laboratories of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Takemi
C
C
Yamamoto
S
S
Tamagake
K
K
Imaoka
S
S
Funae
Y
Y
Kataoka
H
H
Shinoda
S
S
Narimatsu
S
S
D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics*; D019389:Cytochrome P-450 CYP2D6 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D005998:Glycine / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity / Q000235:genetics
D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D019389:Cytochrome P-450 CYP2D6; D005998:Glycine