Transcarboxylase: Strucuture, Flexibility and Mechanism

转羧化酶（Transcarboxylase， TC）是一种大型的多酶复合物，它催化二氧化碳从甲基丙二酰辅酶a转移到丙酮酸，形成草酰乙酸和丙酰辅酶a。该提案的主要目标是提供溶液中TC的结构信息和在不同亚基中发现的两类羧基化位点的详细信息。由于这两种羧基转移半反应与生物医学上重要的（去）羧化酶（如丙酰辅酶a羧化酶和丙酮酸羧化酶）的化学性质相似，因此了解转羧化酶活性位点的机制对分析与疾病状态有关的羧化酶的结构-功能有价值。转羧酶由3种不同的亚基（12S、5S和1.3S）和30条多肽链组成，总分子量为120万道尔顿。由于亚基已经被克隆，并且可以重新组装，并且由于稳定的底物-亚基复合物可以形成，TC为研究大型低聚酶复合物的结构，组装和功能提供了难得的机会。为了利用这一重要机会，该提案设定了三个主要目标：1。探讨12S活性位点的化学性质及其作用机制。拉曼差分光谱（主要是12S单晶）将用于定义底物结合时底物和活性位点基团的变化。2. 探讨5S和6S活性位点的化学性质及其作用机理。拉曼差分光谱将用于探测基材-5S相互作用的细节，其中涉及5S单晶的研究将发挥主要作用。3. 利用荧光共振能量转移绘制全息TC的亚基间和活性位点-活性位点距离。

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.