Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.
1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.
Maekawa
K
K
Project Team for Pharmacogenetics, National Institute of Health Sciences, Tokyo, Japan.
Yoshimura
T
T
Saito
Y
Y
Fujimura
Y
Y
Aohara
F
F
Emoto
C
C
Iwasaki
K
K
Hanioka
N
N
Narimatsu
S
S
Niwa
T
T
Sawada
J
J
D000483:Alleles; D000818:Animals; D002220:Carbamazepine / Q000378:metabolism*; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002220:Carbamazepine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D008874:Midazolam