Structure and Mechanism of Yeast Pyruvate Decarboxylase

描述（由申请人提供）：本研究的总体目标是大大提高我们对结构-功能关系的理解，包括酵母丙酮酸脱羧酶的机制和调控。这种酶需要硫胺素二磷酸（维生素B1辅酶）和脱羧丙酮酸在酒精发酵的倒数第二步。下一个时期需要资金继续对这种酶进行研究，主要目标如下：（1）利用各种光谱方法进一步研究催化残基的作用。通过使用中间体分配和快速淬灭方法直接评估所有关键的二磷酸硫胺素结合中间体的浓度，确定所有活性中心变体以及野生型酶中的限速步骤;（2）实验旨在确定PI及其同事针对该酶提出的“功能性二聚体中的交替活性位点”机制的结构起源和途径，特别是通过识别催化中心之间的信号转导途径。实验的目的是确定的结构起源和途径的二聚体-二聚体相互作用，导致两种不同的构象观察到的X-射线研究。特别是，Pl的小组最近报道了动力学研究，表明有两种活性构象的酶，一个受底物调节，另一个不是。重要的是进一步描绘这两种类型构象的结构基础，并且那些可以结晶的结构将在与匹兹堡大学医学院William Furey的持续合作中接受高分辨率X射线方法。目标1和2将严重依赖于PI不仅引入任何所需取代，而且产生具有任何所需组成的修饰亚基的四聚体的能力。这种酶继续提供一个杰出的范例，不仅为硫胺素依赖性酶，而且为同源四聚体酶的活性中心的相互作用。

DESCRIPTION (provided by applicant): The overall objective of this research is to very much enhance our understanding of structure-function relationships, including the mechanism and regulation of the yeast pyruvate decarboxylase. This enzyme requires thiamin diphosphate (the vitamin B1 coenzyme) and decarboxylates pyruvate in the penultimate step of alcohol fermentation. Funding is requested for the next period to continue research on this enzyme with the following major goals: (1) Further studies of the role of catalytic residues using a variety of spectroscopic methods. Determination of the rate-limiting steps in all active center variants, as well as wild-type enzyme, by direct assessment of the concentration of all key thiamin diphosphate-bound intermediates using intermediate partitioning and rapid-quench methods; (2) Experiments designed to identify the structural origins of and pathway for the 'alternating active sites in a functional dimer' mechanism proposed by the PI and coworkers for this enzyme, specifically by identifying the signal transduction pathway between catalytic centers. Experiments are designed to identify the structural origins of and pathway for dimer-dimer interactions leading to the two different conformations observed by the X-ray studies. In particular, the Pl's group has recently reported kinetics studies indicating that there are two active conformations of the enzyme, one regulated by substrate, the other is not. It is important then to further delineate the structural basis for these two types of conformations and those constructs that can be crystallized will be subjected to high-resolution X-ray methods in an ongoing collaboration with William Furey, Univ. of Pittsburgh, School of Medicine. Goals 1 and 2 will be heavily dependent on the Pl's ability to not only introduce any desired substitution, but also to create tetramers with any desired composition of modified subunits. This enzyme continues to provide an outstanding paradigm not only for thiamin-dependent enzymes, but also for interaction of active centers in homo-tetrameric enzymes.