To evaluate CYP2D6 mutants as drug-metabolizing enzymes, we expressed four single amino acid-substituted mutants (P34S, G42R, R296C and S486T) and two double amino acid-substituted mutants (P34S/S486T and R296C/S486T) in yeast cells. The profiles of P34S, G42R and P34S/S486T in microsomal expression levels and oxidation capacities towards debrisoquine (DB) and bunitrolol (BTL) were much different from those of the wild type, while the behavior of R296C, S486T and R296C/S486T was similar to the wild type in these indices. CYP contents in microsomal and whole cell fractions, which were measured spectrophotometrically and immunochemically, indicated that a part of P34S protein was located in microsomal membranes whereas most of P34S protein was in cytosolic fraction. Enzyme assay revealed that P34S showed much lower activities in DB and BTL 4-hydroxylations than the wild type in terms of nmol/min/mg protein, but similar activities to the wild type in terms of nmol/min/nmol CYP, indicating that P34S, which can anchor to endoplasmic reticulum membrane and hold a prosthetic heme group, retains normal functions. However, G42R did not have normal functions even in terms of nmol/min/nmol CYP; i.e., its Km values for DB and BTL oxidations were much higher than those of the wild type. The results indicate that similar to the substitution of Pro-34 to Ser, that of Gly-42 to Arg results in the decrease in the enzymatic function but in a manner different from P34S.
