Structure-function analysis of human cytochrome P450 3A4 using 7-alkoxycoumarins as active-site probes

摘要

The oxidation of a series of seven alkyl ethers of 7-hydroxycoumarin by cytochrome P450 3A4 (CYP3A4) has been studied to probe the active site of the enzyme. TLC of the reaction mixture showed formation of metabolites other than 7-hydroxycoumarin, The separation and characterization of the different metabolites of the C4 to C7 compounds were achieved using a combination of TLC, HPLC, and gas chromatography-electron impact mass spectra, Among the 7-alkoxycoumarins, 7-hexoxycoumarin was found to be the most suitable candidate for investigating the active site of cytochrome CYP3A4, due to the well-separated metabolite peaks on TLC and HPLC, 7-Hexoxycoumarin was found to produce three side-chain hydroxylated products besides 7-hydroxycoumarin: 7-(5-hydroxyhexoxy)coumarin, 7-(4-hydroxyhexoxy)coumarin, and 7-(3-hydroxycoumarin), The substitution of residues from substrate recognition sites -1, -4, -5, and -6 of CYP3A4 showed a strong influence on the product profile of 7-hexoxycoumarin, the most prominent effects observed with mutants at residues 119, 301, 305, 370, 373, and 479, The docking of 7-hexoxycoumarin into a molecular model of CYP3A4 also confirmed the presence of these residues within 5 A of the substrate. A comparative study of cytochrome P450 2B1 showed that the active-site mutants F206L, T302V, V363A, and 54780 but not V363L exhibited a dramatic decrease in total 7-hexoxycoumarin hydroxylation, The study suggests that although the electronic nature of the substrate is important, enzymatic constraints significantly contribute to CYP3A4 selectivity. (C) 2000 Academic Press. [References: 42]