ENZYMES THAT DECARBOXYLATE PYRUVATE

这一建议；L关注剩余结构的划定 和丙酮酸脱羧酶的机理问题(PDC，E.C.4.1.1.1)， 可能是最简单的需要硫胺素的非氧化脱羧酶 二磷酸(ThDP)，维生素B1辅酶，在 人类新陈代谢)。酿酒人的酵母酶被提纯为完全活性 α4和β4同分异构体和α4结构通过以下方式结晶 匹兹堡退伍军人医院的人工智能和合作者。一个2.4电子 该酶的密度图谱已完成95%以上，包括 辅酶结合部位。这张地图已经在手中，这是第一次 在这一大群α-酮酸脱羧酶的代表上， 可以设计合理的实验来探索：a. 酶结合辅酶在没有底物的情况下，以及在一些 三种假定的共价底物-辅酶络合物；B. 催化活化(在没有底物和存在底物的情况下 两个芳香环(噻唑和4-氨基吡啶)的活化)， 以及对两个环(不仅是噻唑环)都是 参与催化；C.辅酶的环境和 例如，它周围的氨基酸在催化中的作用 确定周围的疏水氨基酸是否提供了一种“溶剂” 从而降低了多个键的过渡态能量 步骤，如模型研究所建议；以及D.结构基础和 底物活化的结构和机理后果 (除其他工具外，还使用丙酮胺，一种非脱羧基物质 能够将酶从低水平转换为高水平的替代物 活动表格)。在解决这些问题的工具中最突出的 将是(在撰写时所有这些都在手中)：a.x射线 结晶学方法(这部分研究是合作的 与富瑞、萨克斯和退伍军人医院生物晶体学同事的合作 实验室/大学B.发现氨基酸的定点突变 基于x射线结晶学的催化和调节中心附近 和机理信息；C.进一步阐明化学和 ThDP底物之一的烯胺的酶结合环境 PDC上的共价络合物；4.基于x射线坐标的模拟 帮助改进机械模型，帮助设计更有洞察力 实验，并比较两种酶的三维模型 与PDC具有很高的序列同源性(丙酮酸氧化酶和 乙酰乳酸合成酶)，通过丙酮酸具有相同机制 脱羧基，但此后出现显著分歧。毫无疑问， 拟议的研究将解决的基本问题包括 对从事以下工作的许多其他研究小组具有深刻的兴趣和意义 硫胺素遍布全球。

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.