Structure-function analysis of human CYP3A4 using a specific proinhibitory antipeptide antibody.
An anti-peptide antibody targeted against residues 253 to 269 of human CYP3A4 was produced that specifically and potently inhibited its activity in human hepatic microsomal fraction (>90%). The function of this region in P450 catalysis was investigated. Antibody binding to CYP3A4 was unable to affect the magnitude of the Type I spectrum on addition of testosterone. It also had no effect on the K(m) of the enzyme for testosterone, but it did cause a marked decrease in V(max) (>90%) of testosterone 6 beta-hydroxylation. There was no change in the ability of the antibody-bound CYP3A4 to form the steady-state level of the enzymatically or chemically reduced P450-CO complex or even the steady-state level of the dioxy-ferrous complex during testosterone metabolism, but the oxidation of NADPH by CYP3A4 in the presence of antibody was 60% that of CYP3A4 in the absence of antibody. The binding of the antibody also resulted in potent inhibition of cumene hydroperoxide-supported testosterone 6 beta-hydroxylase activity of human liver microsomal fraction (>90%). Our conclusion is that the loop region targeted in CYP3A4 is not involved in substrate binding, in reductase binding, in the transfer of the first or second electron from the reductase to CYP3A4, or in the binding of molecular oxygen. We speculate that antibody binding to CYP3A4 inhibits enzyme activity by destabilizing the ternary hydroperoxo complex, by interfering with the second proton transfer, and/or by interfering with the conformational changes that are suggested to be induced by substrate binding.
Schulz-Utermoehl
T
T
Department of Drug Metabolism, Novo Nordisk A/S, Maaloev, Denmark. tisu@novo.dk
Mountfield
R J
RJ
Bywater
R P
RP
Madsen
K
K
Jørgensen
P N
PN
Hansen
K T
KT
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001666:Binding Sites, Antibody; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010455:Peptides / Q000276:immunology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human