CRYSTAL STRUCTURE OF A TRANSCARBOXYLASE (TC) MULTIENZYME COMPLEX

转羧酶 (TC) 多酶复合物的晶体结构

• 批准号: 8169332
• 负责人: VIVIEN YEE
• 金额: $ 0.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169332
• 关键词: Active Sites; Biochemical; Biological Models; Biotin; Catalysis; Complex; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Funding; Goals; Grant; Human; Institution; Ligands; Metabolic; Metabolic Diseases; Methods; Methylmalonyl-CoA carboxyltransferase; Molecular; Multienzyme Complexes; Mutation; Proteins; Publishing; Regulation; Research; Research Personnel; Resources; Source; Structure; United States National Institutes of Health; base; dalton; design; dimer; enzyme mechanism; insight; polypeptide; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of the proposed research is to determine crystal structures of the transcarboxylase (TC) multienzyme complex and related proteins. TC is a 1.2 million dalton biotin-dependent bacterial metabolic enzyme that has long served as a model system for human biotin-dependent metabolic enzymes which have been difficult to study using biochemical and structural methods; none of the human or mammalian biotin-dependent enzymes have been crystallized. Crystal structures of TC will provide insight into mechanisms of assembly and catalysis for biotin-dependent metabolic enzymes. Intact 26S TC contains 30 polypeptide chains of three types: the catalytic 12S which forms a hexamer, the catalytic 5S which forms a dimer, and the biotinylated 1.3S which shuttles between the 12S and 5S active sites. The 26S TC can lose several 5S subunits to give the 24 polypeptide chain 18S, which is also enzymatically active. We have recently published crystal structures of the isolated 12S subunit (1) and of the isolated 5S subunit (2), in free form and as several active site complexes, determined using synchrotron radiation. While these structures provide some initial understanding of enzyme mechanism, structures of the 18S and 26S subunits are expected to be much more informative in order to understand multienzyme complex assembly and regulation, as well as to extend speculations about enzyme mechanism and the molecular basis of human metabolic disorders resulting from mutations in the human enzymes. We propose to determine crystal structures of TC 26S, 18S and 6S, which are hetero-oligomeric forms containing 3 or 2 different subunits, as well as additional structures of isolated 12S and 5S, either as active site ligand complexes or mutations designed to probe enzyme mechanism or to reproduce the effect of human mutations identified in metabolic diseases.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构为 研究中心，而研究中心不一定是研究者所在的机构。 该研究的目的是确定转羧酶（TC）多酶复合物和相关蛋白质的晶体结构。TC是一种120万道尔顿的生物素依赖性细菌代谢酶，长期以来一直作为人类生物素依赖性代谢酶的模型系统，这些生物素依赖性代谢酶很难使用生物化学和结构方法进行研究;人类或哺乳动物的生物素依赖性酶都没有结晶。TC的晶体结构将为生物素依赖性代谢酶的组装和催化机制提供深入了解。完整的26 S TC含有三种类型的30条多肽链：形成六聚体的催化12 S，形成二聚体的催化5S，以及在12 S和5S活性位点之间穿梭的生物素化1.3S。26 S TC可以失去几个5S亚基，以产生24多肽链18 S，其也具有酶活性。我们最近发表了分离的12 S亚基（1）和分离的5S亚基（2）的晶体结构，以游离形式和作为几个活性位点复合物，使用同步辐射测定。虽然这些结构提供了一些酶机制的初步了解，18 S和26 S亚基的结构预计将是更多的信息，以了解多酶复合物的组装和调节，以及扩展有关酶的机制和人类代谢紊乱的分子基础的推测，导致在人类酶的突变。我们建议确定TC 26 S，18 S和6S的晶体结构，这些晶体结构是含有3或2个不同亚基的异源寡聚体形式，以及分离的12 S和5S的额外结构，无论是作为活性位点配体复合物或突变，设计用于探测酶机制或再现代谢疾病中鉴定的人类突变的影响。