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首页> 外文学位 >A tale of two enzymes: Identification of an unknown ligand bound to cytochrome P450 2A13 and understanding substrate selectivity of cytochrome P450 2E1.
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A tale of two enzymes: Identification of an unknown ligand bound to cytochrome P450 2A13 and understanding substrate selectivity of cytochrome P450 2E1.

机译：两种酶的故事：鉴定与细胞色素P450 2A13结合的未知配体，并了解细胞色素P450 2E1的底物选择性。

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摘要

Cytochrome P450 (CYP) is the predominate superfamily of enzymes responsible for Phase I metabolism of drugs and other xenobiotics. Understanding the structural reasons for the substrate selectivity of these enzymes is important for both pharmacological and toxicological reasons. Two isoforms of interest from this enzyme superfamily that are CYP2A13 and CYP2E1.;Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC--UV/vis--MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was unambiguously identified as unsubstituted indole, which facilitated refinement of two alternate conformations of indole in the CYP2A13 crystal structure active site.;Human cytochrome P450 2E1 (CYP2E1) is a xenobiotic metabolizing enzyme that is highly conserved among mammals. In addition to small molecular weight exogenous drugs like the analgesic acetaminophen and the volatile anesthetic halothane, CYP2E1 is also involved in endogenous fatty acid metabolism. To more fully understand the structural factors that contribute to the substrate selectivity of CYP2E1, it has been cocrystallized with two structurally different heme-binding compounds: indazole, a small molecular weight inhibitor and o-imidazolyl-decanoic acid, a fatty acid analog. Comparison of the CYP2E1 structures shows that only small side chain movements are required for the accommodation of the much larger fatty acid analog. Rotation of the side chain of F298 causes a change in the active site volume from 190 A3 in the indazole-bound structure to 440 A3 in the o-imidazolyl-decanoic acid-bound structure. Future work will be focused on cocrystal structures of CYP2E1 with both longer and shorter chain analogs to better understand the ability of the enzyme to metabolize a variety of fatty acids substrates.
机译：细胞色素P450（CYP）是负责药物和其他异源生物的I期代谢的主要酶超家族。出于药理和毒理学原因，了解这些酶的底物选择性的结构原因很重要。 CYP2A13和CYP2E1是该酶超家族的两个感兴趣的同工型;细胞色素P450 2A13（CYP2A13）是一种肺特异性酶，已知会激活强效烟草致癌物4-（甲基亚硝基氨基）-1-（3-吡啶基）-1-丁酮（NNK）成两种致癌代谢物。 CYP2A13已结晶，X射线衍射实验阐明了该酶的结构，但酶活性位点中存在未知配体。该未知配体被怀疑是吲哚，但必须开发一种选择性方法来区分蛋白质样品中的吲哚及其代谢产物。我们成功地修改了微生物比色法，通过对二甲基氨基肉桂醛（DMACA）的衍生化，以分光光度法区分了纳摩尔浓度下的吲哚和许多可能的吲哚代谢物。利用DMACA偶联产生的生色团作为HPLC检测的UV标记，通过质谱（HPLC-UV / vis-MS / MS）对吲哚进行了进一步区分。结晶蛋白中的配体被明确鉴定为未取代的吲哚，这有助于精化CYP2A13晶体结构活性位点中两个交替构象的吲哚。人细胞色素P450 2E1（CYP2E1）是一种在哺乳动物中高度保守的异种生物代谢酶。除小分子量外源性药物如镇痛药对乙酰氨基酚和挥发性麻醉药氟烷外，CYP2E1还参与内源性脂肪酸代谢。为了更全面地了解有助于CYP2E1的底物选择性的结构因素，它已与两种结构上不同的血红素结合化合物共结晶：吲唑（一种小分子量抑制剂）和邻咪唑基-癸酸（一种脂肪酸类似物）。 CYP2E1结构的比较表明，容纳较大的脂肪酸类似物仅需较小的侧链运动。 F298侧链的旋转导致活性部位的体积从与吲唑结合的结构中的190 A3变为与邻咪唑基与癸酸结合的结构中的440 A3。未来的工作将集中在CYP2E1与长链和短链类似物的共晶体结构上，以更好地了解该酶代谢各种脂肪酸底物的能力。

著录项

  • 作者

    Porubsky, Patrick R.;

  • 作者单位

    University of Kansas.;

  • 授予单位 University of Kansas.;
  • 学科 Chemistry Biochemistry.
  • 学位 M.S.
  • 年度 2009
  • 页码 125 p.
  • 总页数 125
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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