Effects of G169R and P34S substitutions produced by mutations of CYP2D6*14 on the functional properties of CYP2D6 expressed in V79 cells.

CYP2D6 is a polymorphic enzyme that catalyzes the oxidation of various drugs. At least 40-mutant alleles of CYP2D6 have been reported. CYP2D6*14, which is one of them found in Asian populations, causes deficient activity of CYP2D6. Four amino acid substitutions, P34S, G169R, R296C, and S486T, are present in the protein encoded by CYP2D6*14 (CYP2D6 14). Among them, G169R is thought to be a definitive substitution because it is unique to CYP2D6 14. However, a previous study showed that the activity of G169R-substituted CYP2D6 was about 40% of wild-type CYP2D6, suggesting that a combination of G169R and other substitutions may be required to abolish the activity of CYP2D6. In the present study, we examined the effects of combined substitutions of G169R and P34S on the functional properties of CYP2D6 and compared them with those of a single substitution of G169R or P34S using a cDNA expression system of V79 cells. The results showed that a combined substitution of G169R and P34S reduced the activities of CYP2D6 to less than the detection limit of our analytical method for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation. However, these activities were not completely abolished by a single substitution of P34S or G169R. The findings suggest that simultaneous substitution of G169R and P34S is crucial for almost completely abolishing the activity of CYP2D6 at least in V79 cells, although whether the absence of metabolism is due to the absence of functional protein or catalytic incompetency remains unclear because the levels of CYP2D6 protein expressed in V79 cells were too low to be determined by difference CO-reduced spectra.