Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily.
Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144-->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359-->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in the amino acid composition of these 2C9/10 proteins can affect the functional specificities towards both tolbutamide and phenytoin.
Veronese
M E
ME
Department of Clinical Pharmacology, Flinders University of South Australia, Bedford Park.
Doecke
C J
CJ
Mackenzie
P I
PI
McManus
M E
ME
Miners
J O
JO
Rees
D L
DL
Gasser
R
R
Meyer
U A
UA
Birkett
D J
DJ
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D013426:Sulfaphenazole / Q000494:pharmacology; D014044:Tolbutamide / Q000378:metabolism; D014162:Transfection
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D013426:Sulfaphenazole; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; C053553:tolbutamide 4-hydroxylase