Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 164-182).

Human glutathione synthetase (hGS) is a homodimeric enzymes that catalyzes the second step in the biological synthesis of glutathione, a critical cellular antioxidant. The enzyme exhibits negative cooperativity towards the γ-glutamylcysteine (γ-GC) substrate. In this type of allosteric regulation, the binding of γ-GC at one active site significantly reduces substrate affinity at a second active site over 40 Å away. The presented work explores protein-protein interactions, substrate binding, and allosteric communication through investigation of three regions of hGS: the dimer interface, the S-loop, and the E-loop. Strong electrostatic interactions across the dimer interface of hGS maintain the appropriate tertiary and quaternary enzymatic structure needed for activity. The S-loop and E-loop of hGS form walls of the active site near γ-GC, with some residues serving to bind and position the negatively cooperative substrate. These strong interactions in the active site serve as a trigger for allosteric communication, which then passes through hydrophobic interactions at the interface. A comprehensive computational and experimental approach relates hGS structure with activity and regulation. ATP-grasp enzymes, including hGS, utilize ATP in the nucleophilic attack of a carboxylic acid in a reaction thought to proceed through the formation of an acylphosphate intermediate. Small metal cations are known to chelate the terminal phosphates of actives site ATP, yet the role of these atoms remains unclear. In the presented work, a computational metal substitution study establishes the role these divalent cations in the catalysis of peptide bonds. The simple model is used to determine the impact of metal cations on the thermodynamics and kinetics, an important stepping stone in understanding the importance of metal cations in larger biological systems.

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The tripeptides glutathione (GSH; γglutamyl-cysteinyl-glycine) and homoglutathione (hGSH; γglutamyl-cysteinyl-β-alanine) are abundant cytosolic tripeptides in legumes. The reactive cysteinyl sulphydryl group enables GSH or hGSH to act as the major cellular redox buffer through the formation of disulphides with other GSH/hGSH molecules. GSH can also form disulphides with cysteinyl groups within proteins, which is termed 5-thiolation, a reversible modification, protecting proteins from irreversible inactivation of thiol residues, as well as being important in regulating protein activity. Following treatment with fungal cell wall elicitors, plant cells produce reactive oxygen species (ROS) which results in cellular oxidative stress. In animal cells ROS generation induces antioxidant defences which include the accumulation of glutathione (GSH) and the formation of mixed disulphides between proteins and GSH. It was hypothesised that following treatment with a fringal elicitor, plant cells also thiolate proteins. It was of interest to determine how protein thiolation changed in response to changes in thiol metabolism known to occur during elicitation, as well as identifying proteins which underwent this modification. Using cell cultures of alfalfa (Medicago saliva L.), a leguminous plant containing both GSH and hGSH, changes in thiol content upon treatment with a fungal cell wall preparation elicitor were determined. By inhibiting protein synthesis and labelling the thiol pools with L-[(^35)S]cysteine, the degree and rate of protein mixed disulphide formation could be monitored in-vivo. To induce the elicitation response, alfalfa cell cultures were treated with a fungal cell-wall elicitor. Following elicitor treatment GSH, but not hGSH, was found to accumulate, with an associated increase in GSH, but not hGSH, forming mixed disulphide with protein. In order to use proteomic tools to identify thiolated proteins, the oxidative stress response in cell cultures of Arabidopsis, a GSH containing species, was then characterised. The level of protein-bound GSH was found to increase following treatment of cell cultures with the oxidant tert-hutyl hydroperoxide and this was associated with changes in cellular thiols. When proteins S-thiolated either in-vivo, or in-vitro, with [(^35)S]-GSH were resolved by SDS-PAGE under non-reducing conditions, a large number of radiolabelled polypeptides were identified in oxidatively stressed preparations. Testing the hypothesis that GSH-dependent enzymes may undergo S-thiolation, proteins which bound GSH were isolated from Arabidopsis using GSH-afFinity chromatography. A number of 30 kDa polypeptides were isolated and found to be S-thiolated under oxidative conditions in-vitro. Several of these were subsequently identified, notably members of the glutathione transferase (GST) superfamily. Representative recombinant GSTs from Arabidopsis, maize and soybean were expressed, Violated in-vitro and the effect on activity determined. Several thiolatable GSTs were identified from Arabidopsis, notably the members of the family of dehydroascorbate reductases (DHAR I, 11, III) and lambda GSTs. Further analysis by elecfrospray mass-spectroscopy confirmed the covalent binding of GSH to DHAR isoenzymes during in-vitro thiolation. It was concluded that S-thiolation of proteins is a commonly observed reversible modification of proteins in plants exposed to oxidative stress with potentially important consequences in cytoprotection and regulation.