S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. Biophysical methods were used to elucidate the mechanism and molecular consequences of S-nitrosation of glutathione transferase (GST) P1-1, a ubiquitous homodimeric detoxification enzyme and important target for cancer therapeutics. Transient kinetic techniques, isothermal titration calorimetry and protein engineering were used to develop a minimal mechanism for S-nitrosation of GSTP1-1, the first for any protein. Cys47 of GSTP1-1 is S-nitrosated according to a conformational selection mechanism, with the chemical step limited by a pre-equilibrium between the open and closed conformations of a dynamic helix at the active site. Cys101, in contrast, is Snitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. S-Nitrosation at Cys47 and Cys101 was found to reduce the detoxification activity of GSTP1-1 by 94%. Circular dichroism spectroscopy, acrylamide quenching and amide hydrogen-deuterium exchange mass spectrometry experiments revealed that the loss of activity is due to the introduction of local disorder at the active site. Furthermore, the modification destabilises domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation, and provide general insight into the mechanism of S-nitrosation and its effect protein stability and dynamics.

Glutathione is one of the most abundant organic compounds found in many biological systems. It is a non-protein tripeptide of Glutamic acid, Cysteine and Glycine. Due to its stability, high cellular concentrations and structural features like gammaglutamyl linkage and sulfhydryl group, glutathione involves with many biological pathways including: cellular defense against xenobiotics and naturally occurring deleterious compounds, like free radicals, metal sequestering, and maintaining cellular sulfhydryl status. Glutathione has been broadly studied however the literature associated with Zn2+ coordination is not clear. This study is focused on collecting thermodynamic data of glutathione binding to Zn2+ metal ions using calorimetric technique. Isothermal titration calorimetry has been recommended as an excellent method to determine the association constant (K), enthalpy change (delta H), and binding stoichiometry (n) of a binding process. These parameters associated with Zn2+ binding glutathione deconvoluted from a series of complex equilibria provide an insight into what drives these reactions forward.

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A new potent competitive inhibitor of glyoxalase II, S-carbobenzoxglutathione (CBG) (Ki=0.065mM) was synthesized. The homogenous enzyme was obtained by a simple two-step CBG-affinity column chromatographic procedure.

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This thesis describes the synthesis and preliminary enzymatic study of glutaryl-S-(8-aminooctyl)-L-cysteinylglycine and glutaryl-S-(10-aminodecyl)-L-cysteinylglycine as inhibitors of glyoxalase I. These analogs of glutathione were prepared as potential ligands for affinity chromatography purification of glyoxalase I. The compounds were synthesized by a seven-step procedure in overall yields of 24% for the octyl analog and 33% for the decyl analog. Both compounds exhibited mixed type inhibition of the enzyme, with the decyl derivative being more inhibitory than the octyl derivative. The inhibition was nonlinear (parabolic) for both compounds. Although less inhibitory than the corresponding S-substituted glutathione derivatives, these analogs are promising candidates for affinity chromatography ligands. Such compounds may also be useful in studying the mechanism of glyoxalase I.

The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again act as partial non-competitive inhibitors of the resensitized enzyme and suggest that only one form of the enzyme is present. The physiological significance of the two enzyme forms is discussed.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 150 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.