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

被引:37
作者
Khan, KK [1 ]
Halpert, JR [1 ]
机构
[1] Univ Texas, Med Branch, Dept Pharmacol & Toxicol, Galveston, TX 77555 USA
关键词
cytochrome P450 3A4; 7-alkoxycoumarin; computer modeling; enzymatic constrains; electronic influence;
D O I
10.1006/abbi.1999.1578
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
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.
引用
收藏
页码:335 / 345
页数:11
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