TRANSCARBOXYLASE, A 12MDA 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。 拟议研究的目标是确定转羧酶(TC)多酶复合体的晶体结构。TC是一种120万道尔顿的依赖生物素的细菌代谢酶，长期以来一直被用作人类依赖生物素的代谢酶的模型系统；人类或哺乳动物的依赖生物素的酶都没有结晶。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：催化六聚体12S、催化二聚体5S和穿梭于12S和5S活性中心之间的生物素化1.3S。26S TC可以失去几个6S(5S+1.3S异构体)亚基而得到18S，这也是酶活性的。我们最近发表了用同步辐射测定的分离的12S和5S亚基的晶体结构。我们建议确定TC 6S、18S和26S的晶体结构，以及分离的12S和5S的额外结构，作为活性位点配体复合体或旨在探索酶机制的突变，或重现代谢性疾病中已发现的人类突变的影响。

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. 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; 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 hexameric 12S, the catalytic dimeric 5S, and the biotinylated 1.3S which shuttles between the 12S and 5S active sites. The 26S TC can lose several 6S (5S+1.3S heterotetramers) subunits to give 18S, which is also enzymatically active. We have recently published crystal structures of the isolated 12S and 5S subunits, determined using synchrotron radiation. We propose to determine crystal structures of TC 6S, 18S, and 26S, 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.