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.

转羧酶（TC）是一种大型多酶复合物，催化CO2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和化学上类似的生物医学上重要的（脱）羧化酶，如丙酰辅酶A羧化酶和丙酮酸羧化酶，转羧酶活性位点的机制的知识将是有价值的结构功能分析的羧化酶参与疾病状态。 转羧酶由12 S、5S和1.3S三种不同亚基和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，并且由于可以形成稳定的底物-亚基复合物，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. 探讨12 S活性中心的化学性质和作用机理。 拉曼差光谱，主要与12 S的单晶，将被用来定义在基板和活性位点基团的基板结合后的变化。 2. 探讨5S和6S活性中心的化学性质和作用机理。 拉曼差光谱将被用来探测基板5S相互作用的细节，与研究涉及单晶5S发挥了重要作用。 3. 利用荧光共振能量转移技术对全息TC的亚基间距离和活性位点间距离进行定位。