PYRUVATE DECARBOXYLASE STRUCTURE/FUNCTION RELATIONSHIPS

这项研究的总体目标是大大扩展我们的 了解一类酶的结构-功能关系， 在新陈代谢中具有根本重要性; α-酮酸脱羧酶。 尽管多年的研究和他们的广泛的重要性，在基础 生物化学，目前还没有一个可用的结构例子 对于任何种类的硫胺素二磷酸（TDP）依赖酶，以及许多 结构相关的问题仍然是关于催化机制， 调控 我们的目标是提供有关基本的详细信息， 亚基（单体）和全酶（四聚体，包括TDP和Mg+2 辅助因子）结构的最广泛研究的TDP依赖性酶 （丙酮酸脱羧酶，PDC）。 这将通过确定3D 全酶复合物的单晶X-射线衍射结构 方法. 有关催化机制和监管的信息将 然后通过比较/对比天然结构与 在存在几种基于机理的抑制剂的情况下的结构和/或 调节化合物。 具体目标是：（1）确定 全酶PDC的结构，使得完整的折叠（链迹） （2）完成并细化结构至2.4 α （3）探讨酶和辅因子在 催化;（4）确定所有辅因子的数量和位置， 并支持/反驳最近关于共同TDP结合的假设 在所有依赖TDP的酶中的位点;（5）鉴定TDP的构象 在有和无底物的情况下;（6）识别和解释 特定氨基酸影响辅因子结合和活性的作用，但 不参与催化;（7）确定参与催化的残基 底物结合和在调控位点的结合;（8）检查 当在反应途径中的各个阶段“冷冻”时的结构;（9） 将结果与相关但功能不同的 酶（转酮醇酶），一旦两个坐标集变得可用。

The overall objective of this research is to substantially extend our knowledge of structure-function relationships in a class of enzymes of fundamental importance in metabolism; the alpha-keto acid decarboxylases. Despite years of study and their widespread importance in basic biochemistry, at present there isn't a single structural example available for thiamin diphosphate (TDP) dependent enzymes of any sort, and numerous structure related questions remain regarding the catalytic mechanism and regulation. Our goal is to provide detailed information about the basic subunit (monomer) and holoenzyme (tetramer, including TDP and Mg+2 cofactors) structures for the most widely studied TDP dependent enzyme (pyruvate decarboxylase, PDC). This will be achieved by determining the 3D structure of the holoenzyme complex via single crystal x-ray diffraction methods. Information regarding the catalytic mechanism and regulation will then be obtained by comparing/contrasting the native structure with the structure in the presence of several mechanism based inhibitors and/or regulating compounds. The specific aims are: (1) to determine the structure of the holoenzyme PDC such that the complete fold (chain trace) can be obtained; (2) to complete and refine the structure to 2.4alpha resolution; (3) to probe the relative roles of the enzyme and cofactors in catalysis; (4) to determine the number of, and locations for all cofactors, and to support/disprove a recent hypothesis regarding a common TDP binding site in all TDP dependent enzymes; (5) to identify the conformation of TDP in the presence and absence of substrate; (6) to identify and explain the roles of specific amino acids affecting cofactor binding and activity, but not involved in catalysis; (7) to identify the residues involved in substrate binding, and binding at the regulatory site; (8) to examine the structure when "frozen" at various stages in the reaction pathway; (9) to compare and contrast the results with a related, but functionally distinct enzyme (transketolase) once both coordinate sets become available.