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 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。列出的是该中心的信息，该中心不一定是研究者所在的机构。 谷胱甘肽 (GSH) 是一种丰富的细胞内三肽，参与氧化应激的保护、半胱氨酸的储存和运输以及半胱氨酸的生物合成。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，并且很大程度上受 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，它们催化 GSH 回收和合成的关键步骤。我们正在结合酶动力学、定点诱变和结构研究这两种酶的结构和功能关系。我们已经成功结晶了酵母谷氨酸半胱氨酸连接酶，并初步将其空间群指定为P41212，我们收集了完整的数据集2.1¿ (Rmerge= 7.1%)并且正在生成重金属衍生物的过程中，我们还获得了GCL的斑马鱼修饰亚基的初步晶体，并试图为催化亚基找到合适的结晶条件以了解变构调节。为了确定必需酶的结构，我们还致力于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。 γ-谷氨酰转肽酶，并获得了 2.0¿对于该项目，我们收集了一种潜在的铅衍生物和两种潜在的汞衍生物的数据集，目前正在尝试确定重金属结合位点，在未来的一年中，我们将投入大量精力来解决每种物质的相问题。未来的工作将集中于使用酶动力学和定点诱变对这些酶进行生化表征，并将以 X 射线晶体学研究提供的结构信息为指导。