COBRE: U NEL: P5: MACROMOLECULAR CRYSTALLOGRAPHY: ENZYM/CRYSTALLOG GSH ENZYMES

COBRE：U NEL：P5：大分子晶体学：酶/晶体 GSH 酶

• 批准号: 7381830
• 负责人: JOSEPH J BARYCKI
• 金额: $ 13.86万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381830
• 关键词: COBRE; NEL; P5; MACROMOLECULAR; CRYSTALLOGRAPHY

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. Glutathione (GSH) is an abundant intracellular tripeptide involved in protection against oxidative stress, storage and transport of cysteine, and the biosynthesis of leukotrienes. Maintenance of intracellular GSH levels is vital to survival of many diverse organisms and is largely controlled by the enzymatic activities of gamma-glutamyl transpeptidase (gGT) and glutamate cysteine ligase (GCL), which catalyze the committed steps in GSH reclamation and biosynthesis respectively. We are investigating structure and function relationships in these two enzymes using a combination of enzyme kinetics, site-directed mutatagenisis, and structural biology. We have successfully crystallized yeast glutamate cysteine ligase, and have tentatively assigned its spacegroup as P41212. We have collected a complete data set to 2.1¿ (Rmerge= 7.1%)and are in the process of generating heavy metal derivatives. We have also obtained preliminary crystals of the zebrafish modifier subunit of GCL and are attempting to find suitable crystallization conditions for the catalytic subunit as well. To understand the allosteric regulation of the essential enzyme, we are also endeavoring to stabilize the complex between the catalytic and regulatory subunits such that the holoenzyme structure can be determined. Concurrently we are pursuing studies of gamma-glutamyl transpeptidase, and have obtained a 2.0¿ dataset. For this project, we have collected data sets on a potential lead derivative and two potential mercury derivatives, and are currently attempting to identify heavy metal binding sites. During the coming year, considerable effort will be placed on solving the phase problem for each of these projects using multiple isomorphous replacement (MIR). Future efforts will focus on biochemical characterizations of these enzymes using enzyme kinetics and site-directed mutagenesis and will be guided by the structural information provided by the x-ray crystallography studies.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。谷胱甘肽（GSH）是一种丰富的细胞内三肽，参与抗氧化应激、半胱氨酸的储存和转运以及白三烯的生物合成。细胞内GSH水平的维持对于许多不同生物体的生存至关重要，并且主要由γ-谷氨酰转肽酶（gGT）和谷氨酸半胱氨酸连接酶（GCL）的酶活性控制，其分别催化GSH回收和生物合成中的关键步骤。我们正在调查的结构和功能的关系，在这两种酶的酶动力学，定点突变，结构生物学的组合。我们成功地结晶了酵母谷氨酸半胱氨酸连接酶，并初步确定其空间群为P41212。我们已经收集了一个完整的数据集到2.1 <$（Rmerge= 7.1%），并正在生成重金属衍生物。我们还获得了初步的晶体的斑马鱼修饰亚基的GCL，并试图找到合适的结晶条件的催化亚基以及。为了了解必需酶的变构调节，我们也在努力稳定催化亚基和调节亚基之间的复合物，以便确定全酶的结构。同时，我们正在进行γ-谷氨酰转肽酶的研究，并获得了2.0的数据集。在这个项目中，我们收集了一个潜在的铅衍生物和两个潜在的汞衍生物的数据集，目前正在试图确定重金属结合位点。在接下来的一年里，将投入大量精力使用多个同晶替代（MIR）来解决这些项目中每个项目的阶段问题。未来的工作将集中在这些酶的生化特性，使用酶动力学和定点诱变，并将由X射线晶体学研究提供的结构信息的指导。