TRANSCARBOXYLASE, A 12MDA MULTIENZYME COMPLEX

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 拟议的研究的目标是确定转羧酶（TC）多酶复合物的晶体结构。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;人类或哺乳动物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：催化六聚体12 S、催化二聚体5S和穿梭于12 S和5S活性位点之间的生物素化1.3S。26 S TC可以失去几个6S（5S+1.3S异源四聚体）亚基以产生18 S，其也具有酶活性。我们最近发表的晶体结构的孤立的12 S和5S亚基，确定使用同步辐射。我们建议确定TC 6S，18 S和26 S的晶体结构，以及分离的12 S和5S的其他结构，无论是作为活性位点配体复合物或突变，旨在探测酶的机制或重现在代谢疾病中鉴定的人类突变的影响。

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.