Functional Assessment of Specific Amino Acid Residues of Cytochrome P4501A1 Using Anti-peptide Antibodies

Abstract Several positively charged amino acid residues in cytochrome P450 have been shown to be involved in the electrostatic association with NADPH-cytochrome P450 reductase. For cytochrome P4501A1, five regions were proposed as the putative binding sites for the reductase (Shen, S., and Strobel, H. W., (1993) Arch. Biochem, Biophys, 304, 257-265). To elucidate the specific roles of each of these amino acid residues, five anti-peptide antibodies defined as 1A, 4A, 5A, 6A, and 7A were generated against these regions containing 8-13 amino acids and were affinity-purified using a peptide-Sepharose 4B column. Analysis by enzyme-linked immunosorbent assay and protein immunoblot techniques demonstrated that three of five anti-peptide antibodies have specific binding to the peptides as well as to cytochrome P4501A1. Incubation of the various anti-peptide antibodies with cytochrome P450 followed by reconstitution with reductase, lipid, and NADPH resulted in significant inhibition of P450 activity for antibodies 5A and 6A, but not for 1A, 4A or 7A.. Antibody 5A also exhibited inhibition of P450 activity supported by cumene hydroperoxide, though the inhibition was 45 to 30% less than the inhibition of reductase-supported activity at each of the increasing concentrations of antibody. Kinetic studies with antibody 5A revealed no change in the K m for the substrate ethoxycoumarin, but rather a dramatic effect on the V max of the cytochrome P4501A1 system whether reconstituted with reductase or supported by cumene hydroperoxide. Characterization of the effects of antibody 5A on cytochrome P4501A1 suggested that the binding of antibody 5A to P4501A1 may change the binding of P4501A1 with reductase. Furthermore, the binding of 5A to cytochrome P4501A1 also lowered the V max of the P450. These results are consistent with the roles for the regions of cytochrome P4501A1 from amino acid residues 269 to 281 (peptide 5) and 454 to 463 (peptide 6) in cytochrome P4501A1 hydroxylation activity.