Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9.
CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.
Tsao
C C
CC
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Wester
M R
MR
Ghanayem
B
B
Coulter
S J
SJ
Chanas
B
B
Johnson
E F
EF
Goldstein
J A
JA
D019943:Amino Acid Substitution / Q000235:genetics; D001189:Aryl Hydrocarbon Hydroxylases; D001216:Asparagine / Q000235:genetics; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D008617:Mephenytoin / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012694:Serine / Q000235:genetics; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D013912:Threonine; D012694:Serine; D006639:Histidine; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D039181:Steroid 16-alpha-Hydroxylase; D008617:Mephenytoin