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.

该主题是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。谷胱甘肽（GSH）是一种涉及抗氧化物胁迫，半胱氨酸的储存和运输以及白细胞生物合成的丰富细胞内三肽。细胞内GSH水平的维持对于许多潜水生物的存活至关重要，并且在很大程度上受到γ-谷氨酰基转肽酶（GGT）和谷氨酸半胱氨酸连接酶（GCL）的酶促活性的控制，该活性分别催化了GSH回收和生物体现的促成步骤。我们正在使用酶动力学，定向的诱变和结构生物学的组合研究这两种酶的结构和功能关系。我们已经成功地结晶了酵母菌谷氨酸半胱氨酸连接酶，并将其空间组分配为P41212。我们已将完整的数据集收集到2.1€（rmerge = 7.1％），并且正在生成重金属衍生物。我们还获得了GCL的斑马鱼修饰符亚基的初步晶体，并正在尝试为催化亚基找到合适的结晶条件。为了了解基本酶的变构调节，我们还努力稳定催化和调节亚基之间的复合物，以便可以确定全酶结构。同时，我们正在研究γ-谷氨基转肽酶的研究，并获得了2.0“数据集”。对于这个项目，我们已经收集了有关潜在铅衍生物和两个潜在汞衍生物的数据集，目前正在尝试鉴定重金属结合位点。在来年，将使用多个同构替换（MIR）为解决这些项目的每个项目的相位问题做出了巨大的努力。未来的努力将使用酶动力学和定向诱变，将重点放在这些酶的生化特征上，并以X射线晶体学研究提供的结构信息为指导。