ENZYMES THAT DECARBOXYLATE PYRUVATE

这个建议;l是关于划定其余的结构 丙酮酸脱羧酶（PDC，E.C.4.1.1.1）的机制问题， 也许是最简单的非氧化脱羧酶需要硫胺素 二磷酸（ThDP，维生素B1辅酶，在维生素B1中具有重要意义， 人体代谢）。 啤酒酵母酶被纯化为完全活性的 α 4和β 4同源四聚体，α 4结构通过 匹兹堡退伍军人医院的人工智能和合作者。 2.4电子 这种酶的密度图超过95%完整，包括 辅酶结合位点。 有了这张地图，第一次 关于这一大群α-酮酸脱羧酶的代表， 可以设计合理的实验来探测：a.的构象 酶结合的辅酶在没有底物的情况下，以及在一些 三种推定的共价底物-辅酶复合物; B. 的 对催化的活化（在不存在和存在底物的情况下 活化）两个芳环（噻唑和4-氨基嘧啶）， 并检验两个环（不仅是噻唑）都是 催化的参与者; c.辅酶的环境和 例如，它周围的氨基酸在催化作用中的功能 确定周围的疏水性氨基酸是否提供“溶剂 效应”，从而降低了一些关键的过渡态能量， 步骤，如模型研究所建议的;以及d.结构基础和 底物活化的结构和机械后果 （除其他工具外，使用非脱羧底物阿伐酰胺， 能够将酶从低水平转变为高水平的替代物 活动形式）。 在解决这些问题的工具中， 将是（所有这些都是“在手”作为书面）：a. x射线 晶体学方法（这部分研究是一个合作项目， Furey、Sax和同事在VA医院的生物组织学 匹兹堡大学实验室）; B.氨基酸定点突变 基于X射线晶体学， 和机械信息; c.进一步阐明化学性质， 三种ThDP底物之一烯胺的酶结合环境 PDC上的共价复合物; 4.基于X射线坐标建模， 帮助完善机械模型，帮助设计更有洞察力的 实验，并比较两种酶的三维模型 与PDC（丙酮酸氧化酶和 乙酰乳酸合成酶），它们通过丙酮酸 脱羧，但此后明显不同。 无疑 拟议的研究将解决的基本问题是 对许多其他研究小组的深刻兴趣和意义， 硫胺素在地球仪中的应用

This proposal;l is concerned with delineation of the remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C.4.1.1.1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewers'yeast enzyme was purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the AI and collaborators at the VA Hospital in Pittsburgh. A 2.4 electron density map of this enzyme is more than 95% complete, including the coenzyme binding site. With this map already in hand, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in some of the three putative covalent substrate-coenzyme complexes; b. the activation towards catalysis (in the absence and presence of substrate activation) of the two aromatic rings (thiazolium and 4-aminopyramidine), and test of the hypothesis that both rings (not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis, for example determining if surrounding hydrophobic amino acids provide a "solvent effect" thereby lowering the transition state energies of a number of key steps, as suggested by model studies ; and d. structural bases and structural as well as mechanistic consequences of substrate activation (using among other tools pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form). Most prominent among the tools to address these questions will be (all of these are "in hand" as of writing): a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh); b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; 4. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to compare the three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research will be of profound interest and significance to many other research groups working on thiamin around the globe.