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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 拟议的研究的目的是确定经羧化酶（TC）多烯酶复合物和相关蛋白的晶体结构。 TC是一种120万个道尔顿生物素依赖性细菌代谢酶，长期以来一直是人类生物素依赖性代谢酶的模型系统，使用生化和结构方法很难研究。人类或哺乳动物依赖性酶均未结晶。 TC的晶体结构将深入了解依赖生物素的代谢酶的组装机理和催化。完整的26S TC包含三种类型的30个多肽链：形成六聚体的催化12s，形成二聚体的催化5s，以及在12s和5s活性位点上穿梭的生物素化的1.3s。 26S TC可能会失去几个5S亚基，从而给出24个多肽链18s，这也具有酶活性。我们最近发表了分离的12S亚基（1）和分离的5S亚基（2）的晶体结构，以自由形式和几种活性位点复合物，使用同步辐射确定。尽管这些结构提供了对酶机制的初步了解，但预计18S和26S亚基的结构将更具信息性，以了解多元素复合物的组装和调节，并扩展有关酶机制和人类enzysemes突变产生的有关酶机制和分子基础的猜测。我们建议确定TC 26s，18s和6s的晶体结构，它们是包含3或2个不同亚基的异性 - 寡聚形式，以及孤立的12s和5s的其他结构，即作为活跃的位点配体配合物或旨在用于探测酶机制的活性位点配体配合物或突变，或者以探测人类突变的效应，以探测人类突变的效应。