Protein-Protein Interactions as Underlying Regulatory Mechanisms of Drug-metabolizing Enzyme Function

Abstract

Cytochrome P450 (P450, CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major drug-metabolizing enzymes known to catalyze substrate oxidation and glucuronidation, respectively. Both enzymes are located within the endoplasmic reticulum (ER) membrane; however, their membrane topologies differ, with P450 facing cytosol and a major part of UGTs located on the luminal side. Because of the large differences in the reactions that they catalyze and their membrane topologies, it had been believed that P450 and UGT function separately. However, some chemicals are oxidized by P450 and undergo further conjugation by UGT. Therefore, it is important to consider that P450 and UGT may form a complex within the ER membrane and regulate each other's function through this interaction. To prove this hypothesis, we constructed a co-expression system for CYP3A4, P450 reductase, and UGT2B7/1A9 with a baculovirus-insect cell expression system. This system allowed us to compare CYP3A4 activity in the presence and absence of UGT2B7/1A9 co-expression and revealed that the UGTs suppress CYP3A4 activity. The suppressive effect of UGT2B7 was not limited to the enzymatic activity of CYP3A4 but also extended to the entire catalytic cycle, which may be resulted from the inhibition of substrate-binding to the P450. Analysis using UGT mutants indicated that one of the hydrophobic regions within the luminal portion of the UGT interacts with CYP3A4. Furthermore, we suggested that UGT1A suppresses CYP3A activity in vivo by treating rats with dexamethasone. Thus, the functional interactions between P450 and UGT would advance our understanding of the large inter-individual differences in drug-metabolizing capacity.