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.
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