CRYSTAL STRUCTURE OF A TRANSCARBOXYLASE (TC) MULTIENZYME COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项拟议研究的目标是确定转羧酶(TC)多酶复合体和相关蛋白质的晶体结构。TC是一种120万道尔顿生物素依赖的细菌代谢酶，长期以来一直被用作人类生物素依赖代谢酶的模型系统，这些酶很难用生化和结构方法进行研究；人类或哺乳动物的生物素依赖酶都没有结晶。TC的晶体结构将对生物素依赖的代谢酶的组装和催化机制提供深入的了解。完整的26S TC含有三种类型的30条多肽链：形成六聚体的催化12S，形成二聚体的催化5S，以及穿梭于12S和5S活性中心之间的生物素化1.3S。26S TC可以失去几个5S亚基，从而得到24个多肽链18S，该多肽链也具有酶活性。我们最近发表了分离的12S亚基(1)和分离的5S亚基(2)的自由形式的晶体结构和几个活性中心的络合物，用同步辐射测定。虽然这些结构提供了对酶机制的一些初步了解，但18S和26S亚基的结构有望提供更多的信息，以便了解多酶复合体的组装和调节，以及扩展对酶机制和人类酶突变导致的代谢疾病的分子基础的推测。我们建议确定TC 26S、18S和6S的晶体结构，它们是包含3个或2个不同亚基的异源低聚形式，以及分离的12S和5S的附加结构，要么作为活性位点配体复合体，要么作为旨在探索酶机制的突变，或者重现在代谢性疾病中发现的人类突变的效应。

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.