Cytochrome P450 1A-ligand interactions: Implications for substrate specificity and inhibitor susceptibility.

摘要

Cytochromes P450 are heme-containing enzymes that are involved in the metabolism of a variety of clinically important drugs, endogenous and exogenous compounds, including a number of procarcinogens. P450 1A subfamily has two members: 1A1 and 1A2. P450 1A1 and 1A2 show high sequence identity (>70%), but display different substrate specificity and inhibitor susceptibility. P450 1A2 is one of the major hepatic P450s, which metabolizes more than 11% of drugs currently on the market. Thus, we focused our attention on studies of this particular P450.;The five key active site residues that are different between P450 1A1 and 1A2 have been proposed to play an important role in determining the substrate binding orientation. We adopted phenacetin, an important substrate marker for P450 1A2, to investigate this role. Kinetic studies have shown that the L382V mutant and other mutants containing the L382V substitution exhibited markedly higher catalytic efficiency than the wild type enzyme, while other four single mutants displayed much lower activity. Stoichiometry studies indicated that the higher coupling occurred due to decreased water formation in the catalytic cycle by L382V and mutants containing the L382V substitution. Docking and molecular dynamic simulations suggested that the L382V substitution enabled the oxidation site of phenacetin to move closer to the ferryl oxygen of heme, thereby promoting phenacetin metabolism.;In order to verify the above mechanism, NMR T1 relaxation measurements were utilized to estimate the distance between protons of phenacetin and ferryl oxygen of oxo-heme of P450 wild type or mutants. The results showed that the time-averaged orientations of phenacetin in the active site were very similar in P450 1A2 wild type and mutants. However, the protons at the site of oxidation of phenacetin were closer to the ferryl oxygen in P450 1A2 L382V and L382V/N312L mutants than P450 1A2 WT, which is consistent with the findings from molecular modeling.;To extend our studies, we explored the interactions between inhibitors and P450 1A2 WT and mutants. Molecular modeling techniques, including docking and molecular dynamic simulations, have been extensively used to predict possible inhibitor-enzyme interactions and describe the docking energy involved. In some cases, for example with residue Phe226, pi-pi stacking might play a major role in these interactions. Good correlations between docking scores and inhibition constants Ki were obtained using AutoDock program.;The combination of molecular modeling and experimental techniques helped us to thoroughly investigate the structure-function relationships of P450 1A2. The insight we gained into the catalytic and inhibition mechanism(s) of this enzyme stresses the importance of the active site topology for P450 activity and provides important implications for the rational design of anticancer drugs.