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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。谷胱甘肽（GSH）是一种丰富的细胞内三肽，参与抗氧化应激、半胱氨酸的储存和运输以及白三烯的生物合成。维持细胞内谷胱甘肽水平对许多不同生物的生存至关重要，并且在很大程度上受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，它们分别催化谷胱甘肽回收和生物合成的承诺步骤。我们正在利用酶动力学、定点诱变和结构生物学的结合来研究这两种酶的结构和功能关系。我们成功地结晶了酵母谷氨酸半胱氨酸连接酶，并初步将其空间基定为P41212。我们已经收集了2.1¿（Rmerge= 7.1%）的完整数据集，并正在生成重金属衍生物。我们还获得了斑马鱼改性剂GCL亚基的初步结晶，并试图为催化亚基寻找合适的结晶条件。为了了解必需酶的变构调节，我们也在努力稳定催化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¿数据集。在这个项目中，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，目前正在试图确定重金属结合位点。在接下来的一年里，我们将投入相当大的努力，利用多重同构替换（MIR）来解决每个项目的阶段问题。未来的工作将集中在利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。

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.