Crystal Structures of the Transcarboxylase Multienzyme Complex and of its Subunits

转羧酶多酶复合物及其亚基的晶体结构

• 批准号: 0401654
• 负责人: Vivien Yee
• 金额: $ 28.72万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0401654&HistoricalAwards=false
• 关键词: Crystal; Structures; Transcarboxylase; Multienzyme; Complex

YeeMCB 0077488The objective of this research is to identify the structural determinants of multienzyme complex formation and biochemical function for transcarboxylase. Transcarboxylase is a 1.2 million dalton multienzyme complex that catalyzes the transfer of CO2 from methylmalonyl coenzyme A to pyruvate, to form propionyl coenzyme A and oxaloacetate, by way of two biotin-dependent half reactions. This multienzyme complex contains three different types of subunits and a total of 30 polypeptide chains: a 396 kDa hexameric central "12S" subunit, six 116 kDa dimeric outer "5S" subunits; and twelve 12 kDa biotinylated "1.3S" linkers. X-ray crystal structures of the holo enzyme and of its largest (12S and 5S) subunits, in free form and bound to substrate or product, will be determined. The first specific aim of this work is to determine the crystal structures of 12S substrate and product complexes, by heavy atom phasing and subsequent molecular replacement methods. These structures will identify the structural determinants of substrate binding and catalysis for the first transcarboxylase half reaction. In particular, the protein residues which interact with substrate or product molecules will be revealed; the geometry and chemistry of these amino acids as well as others nearby will be analyzed in order to speculate about likely catalytic mechanism(s). The second specific aim is to determine the crystal structures of the free 12S protein, and of 12S bound to biotin, by molecular replacement methods. Comparison of these structures with the substrate and product complexes will allow identification of the conformational changes which occur upon substrate or biotin binding, and may extend the structural insight into catalytic mechanism. Finally, the third specific aim is to determine the crystal structures of the 5S, 6S, and holo enzyme forms of transcarboxylase, either free or bound to substrate, by a combination of heavy atom phasing and molecular replacement methods. The 5S structures will provide insight into the catalytic mechanism for the second transcarboxylase half reaction, by identifying amino acids which are important in substrate binding and/or catalysis. The 6S and holo enzyme structures will highlight conformational changes and interaction surfaces which are important for enzyme assembly and function. The assembly of transcarboxylase crystal structures which are being pursued will reveal the features important in homo-oligomeric subunit assembly as well as reveal the structural features of intersubunit assembly. These results will provide a rare structural view of a multienzyme complex. Transcarboxylase has historically served as a model system, which is valuable for understanding the mechanisms for other (de)carboxylase enzymes that carry out similar chemistry.

YeeMCB 0077488本研究的目的是确定转羧酶多酶复合体形成的结构决定因素和生化功能。转羧酶是一个120万道尔顿的多酶复合体，通过两个依赖生物素的半反应，催化二氧化碳从甲基丙二酸辅酶A转移到丙酮酸，生成丙酰辅酶A和草酰乙酸酯。该多酶复合体包含3种不同类型的亚基和30条多肽链：396 kDa六聚体中心“12S”亚基，6个116 kDa二聚体外部“5S”亚基和12 kDa生物素化的“1.3S”连接子。全息酶及其最大亚基(12S和5S)的自由形式和与底物或产物结合的X射线晶体结构将被确定。这项工作的第一个具体目标是通过重原子相变和随后的分子置换方法来确定12S底物和产物络合物的晶体结构。这些结构将确定底物结合的结构决定因素和第一个转羧酶半反应的催化作用。特别是，将揭示与底物或产物分子相互作用的蛋白质残基；将分析这些氨基酸以及附近其他氨基酸的几何和化学，以推测可能的催化机制(S)。第二个具体目标是通过分子置换方法确定游离12S蛋白和与生物素结合的12S蛋白的晶体结构。将这些结构与底物和产物络合物进行比较，将有助于识别底物或生物素结合时发生的构象变化，并可能扩大对催化机理的结构洞察。最后，第三个具体目标是通过重原子相变和分子置换相结合的方法来确定转羧酶的5S、6S和全酶形式的晶体结构，无论是游离的还是与底物结合的。通过识别在底物结合和/或催化中重要的氨基酸，5S结构将为第二个转羧酶半反应的催化机理提供深入的了解。6S和HALO酶结构将突出构象变化和相互作用表面，这对酶的组装和功能是重要的。正在进行的转羧酶晶体结构的组装将揭示同源低聚亚单位组装的重要特征，以及亚单位间组装的结构特征。这些结果将提供一个罕见的多酶复合体的结构视图。转羧酶在历史上一直是一个模型系统，这对于理解其他(去)羧基酶的机制是有价值的，这些酶进行类似的化学作用。