Transcarboxylase: Strucuture, Flexibility and Mechanism

转羧酶：结构、灵活性和机制

• 批准号: 6761798
• 负责人: PAUL R CAREY
• 金额: $ 26.07万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6761798
• 关键词: Raman spectrometry; carboxylation; carboxyltransferase /carbamoyltransferase; chemical kinetics; conformation; crystallization; enzyme complex; enzyme mechanism; enzyme model; enzyme reconstitution; enzyme substrate complex; fluorescence resonance energy transfer; intermolecular interaction; model design /development; molecular site; protein purification; protein structure function; structural biology

Transcarboxylase (TC) is a large multi-enzyme complex that catalyzes the transfer of CO2 from methyl malonyl co-enzyme A to pyruvate to form oxaloacetate and propionyl co-enzyme A. The broad objectives of the proposal are to provide structural information for TC in solution and detailed information on the two classes of carboxylation sites found in separate subunits. Since these two carboxyl transfer half reactions and chemically similar to those for biomedically important (de)carboxylase enzymes, such as propionyl-CoA carboxylase and pyruvate carboxylase, knowledge of mechanism for the transcarboxylase active sites will be of value in structure-function analyses of carboxylases implicated in disease states. Transcarboxylase consists of three different kinds of subunit (12S, 5S and 1.3S) and thirty polypeptide chains with an overall molecular weight of 1.2 million Daltons. Since the subunits have been cloned, and can be reassembled, and since stable substrate-subunit complexes can be f ormed, TC offers a rare opportunity to study the structure, assembly and function of a large oligomeric enzyme complex. To exploit this important opportunity the proposal sets three main goals: 1. To probe the chemistry and mechanism of the 12S active site. Raman difference spectroscopy, principally with single crystals of 12S, will be used to define changes in substrate and active site groups upon substrate binding. 2. To probe the chemistry and mechanism of the 5S and 6S active sites. Raman difference spectroscopy will be used to probe the details of substrate-5S interactions, with studies involving single crystals of 5S playing a major role. 3. To map inter-subunit and active site-active site distances in holo TC using fluorescence resonance energy transfer.

经羧化酶（TC）是一种大型的多酶复合物，可催化二氧化碳从丙二酰二酶A到丙酮酸的转移，形成丙酮酸，形成草乙酸和丙酰辅酶A.该建议的广泛目标旨在为carbober carboxyl carboxyl sere carbober inters提供结构信息。 由于这两种羧基转移一半反应，并且在化学上与生物医学重要的（DE）羧化酶（例如丙酰辅酶A羧化酶和丙酮酸羧化酶）的化学相似，因此，羧化酶活性位点的机制知识在与疾病状态的羧基结构 - 功能分析中具有价值具有价值。 转羧酸酶由三种不同类型的亚基（12s，5s和1.3s）和三十多肽链组成，总分子量为120万达尔顿。 由于亚基已被克隆，并且可以重新组装，并且由于稳定的底物 - 亚基配合物可以被填充，因此TC提供了一个难得的机会来研究大型寡聚酶复合物的结构，组装和功能。 为了利用这一重要机会，该提案设定了三个主要目标：1。探究12S活跃部位的化学和机制。 拉曼的差异光谱法主要具有12s的单晶，将用于定义底物结合后底物和活性位点组的变化。 2。探测5s和6s活性位点的化学和机制。 拉曼差异光谱法将用于探测底物5s相互作用的细节，其中涉及5s的单晶的研究起着主要作用。 3。使用荧光共振能量转移绘制Holo TC中的亚基间和主动位点距离。