Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts.
The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.
Department of Pharmacology, School of Medicine, Keio University, Tokyo.
T
T
Yasumori
Y
Y
Yamazoe
R
R
Kato
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000201:enzymology*; D008617:Mephenytoin; D008969:Molecular Sequence Data; D010957:Plasmids; D011110:Polymorphism, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D008617:Mephenytoin