Transcarboxylase: Strucuture, Flexibility and Mechanism

转羧酶(TC)是一个大型的多酶复合体，催化二氧化碳从丙二酸甲酯转移到丙酮酸，生成草酰乙酸酯和丙酰辅酶A。本提案的主要目的是提供TC在溶液中的结构信息和在不同亚基中发现的两类羧化位点的详细信息。由于这两个羧基转移半反应在化学上类似于生物医学上重要的(去)羧基酶，如丙酰辅酶A羧基酶和丙酮酸羧基酶，了解转羧基酶活性部位的机制将在疾病状态下的羧基酶的结构和功能分析中有价值。转羧酶由三种不同的亚基(12S、5S和1.3S)和30个多肽链组成，总分子量为120万道尔顿。由于亚基已经被克隆，并且可以重组，而且可以形成稳定的底物-亚基复合体，TC为研究大分子寡聚体酶复合体的结构、组装和功能提供了难得的机会。为了利用这一重要机会，该提议设定了三个主要目标：1.探索12S活性部位的化学和机制。拉曼差分光谱，主要是12S单晶，将被用来确定底物结合时底物和活性位基团的变化。2.探讨5S和6S活性中心的化学组成和作用机制。拉曼差分光谱将用于探测衬底-5S相互作用的细节，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技术绘制全息甘油三酯的亚基间距离和活性中心-活性中心距离。

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.