Chan W K, Sui Z, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Chem Res Toxicol. 1993 Jan-Feb;6(1):38-45. doi: 10.1021/tx00031a006.
Reaction of cytochrome P450 enzymes with arylacetylenes results in heme N-alkylation [e.g., Komives, E. A., and Ortiz de Montellano, P. R., (1985) J. Biol. Chem. 260, 3330-3336] and/or protein modification [e.g., Gan, L.-S. L., Acebo, A. L. and Alworth, W. L. (1984) Biochemistry 23, 3827-3836]. To clarify the factors that determine whether heme or protein alkylation occurs, we have investigated the cytochrome P450 1A1-catalyzed oxidation of 1-ethynylpyrene (1-EP) and phenylacetylene (PA). Cytochrome P450 1A1 in microsomes from beta-naphthoflavone-induced rats is inactivated in a time- and NADPH-dependent manner by 1-EP and PA. Parallel loss of the heme chromophore is observed with PA but not with 1-EP, although partial heme chromophore loss is observed when the purified, reconstituted enzyme is inactivated by either agent. Product analysis shows that 1-EP and PA are oxidized to, respectively, (1'-pyrenyl)-acetic and phenylacetic acids. In contrast to the inactivation of cytochrome P450 2B1 by PA, no isotope effect is observed on enzyme inactivation or metabolite formation when the acetylenic hydrogen is replaced by deuterium in either 1-EP or PA. Inactivation of cytochrome P450 1A1 by 1-EP results in covalent binding of 0.8-0.9 equiv (relative to total cytochrome P450 content) of the inhibitor to the microsomal protein. The results demonstrate that a single isozyme can be inactivated, depending on the structure of the arylacetylene, by heme or protein alkylation. Spectroscopic binding constants (Ks) show that 1-EP binds to the enzyme with > 2000 times greater affinity that PA.(ABSTRACT TRUNCATED AT 250 WORDS)
细胞色素P450酶与芳基乙炔反应会导致血红素N-烷基化[例如，Komives, E. A., 和 Ortiz de Montellano, P. R., (1985) J. Biol. Chem. 260, 3330 - 3336]和/或蛋白质修饰[例如，Gan, L.-S. L., Acebo, A. L. 和 Alworth, W. L. (1984) Biochemistry 23, 3827 - 3836]。为了阐明决定发生血红素或蛋白质烷基化的因素，我们研究了细胞色素P450 1A1催化的1-乙炔基芘(1-EP)和苯乙炔(PA)的氧化反应。β-萘黄酮诱导的大鼠微粒体中的细胞色素P450 1A1被1-EP和PA以时间和NADPH依赖性方式失活。PA会导致血红素发色团平行丧失，而1-EP则不会，不过当纯化的重组酶被任何一种试剂失活时，会观察到部分血红素发色团丧失。产物分析表明，1-EP和PA分别被氧化为(1'-芘基)-乙酸和苯乙酸。与PA使细胞色素P450 2B1失活不同，当1-EP或PA中的乙炔氢被氘取代时，在酶失活或代谢产物形成方面未观察到同位素效应。1-EP使细胞色素P450 1A1失活导致0.8 - 0.9当量(相对于细胞色素P450总含量)的抑制剂与微粒体蛋白共价结合。结果表明，取决于芳基乙炔的结构，单一同工酶可通过血红素或蛋白质烷基化失活。光谱结合常数(Ks)表明，1-EP与该酶的结合亲和力比PA高2000倍以上。(摘要截短于250字)
