Structure-function analysis of human CYP3A4 using a specific proinhibitoryantipeptide antibody

An anti-peptide antibody targeted against residues 253 to 269 of human CYP3 A4 was produced that specifically and potently inhibited its activity in hu man hepatic microsomal fraction (> 90%). The function of this region in P45 0 catalysis was investigated. Antibody binding to CYP3A4 was unable to affe ct the magnitude of the Type I spectrum on addition of testosterone. It als o 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. T here 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 compl ex or even the steady-state level of the dioxyferrous complex during testos terone 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-sup ported testosterone 6 beta-hydroxylase activity of human liver microsomal f raction (> 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 bi nding of molecular oxygen. We speculate that antibody binding to CYP3A4 inh ibits 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 bindi ng.