Boysen Gunnar, Scarlett Cameron O, Temple Brenda, Combs Terry P, Brooks Natasha L, Borchers Christoph H, Swenberg James A
Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, NC 27599-7431, USA.
Chem Biol Interact. 2007 Mar 20;166(1-3):170-5. doi: 10.1016/j.cbi.2007.01.007. Epub 2007 Jan 17.
1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.
1,3 - 丁二烯主要通过细胞色素P450 2E1代谢生成几种被认为具有毒性和致癌性的环氧化物。丁二烯代谢的第一步是氧化生成1,2 - 环氧 - 3 - 丁烯（EB），这是一种活性代谢产物。研究表明，细胞色素P450可通过活性代谢产物与蛋白质或血红素的共价结合而失活。分子剂量学研究清楚地表明，丁二烯代谢遵循超线性剂量反应，提示代谢活化存在饱和现象。在本研究中，确定并模拟了人细胞色素P450 2E1中EB的潜在结合位点，以测试EB是否与对酶活性重要的残基发生共价结合。将市售的人细胞色素P450 2E1与EB反应，用胰蛋白酶消化，然后通过基质辅助激光解吸/电离串联飞行时间质谱（MALDI - MS）分析所得肽段。通过基质辅助激光解吸/电离串联质谱（MALDI - MS/MS）测序确认了EB修饰肽段的身份。结果表明，EB与四个组氨酸和两个酪氨酸残基结合。所有修饰位点均由至少两个相邻且至少八个肽特异性片段确定。蛋白质建模显示，其中两个共价修饰（His(109)，His(370)）与活性位点明显相关，其α碳原子距离已知抑制剂结合位点小于9埃。此外[His(370)]的侧链距离血红素基团在4埃以内，预计其修饰会影响血红素的取向。Tyr(71)的α碳原子距离潜在抑制剂结合位点在14埃以内，且在配体结合时发生构象变化的侧翼区内7埃以内，这可能使Tyr(71)在底物进入和离开活性位点时靠近底物。这些数据支持了EB可通过共价修饰使细胞色素P450 2E1失活的假说，从而为丁二烯代谢增加了一种额外的调节机制。
Zotero 插件