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Metals in Chemistry and Biology: Computational Chemistry Studies

Description: Numerous enzymatic reactions are controlled by the chemistry of metallic ions. This dissertation investigates the electronic properties of three transition metal (copper, chromium, and nickel) complexes and describes modeling studies performed on glutathione synthetase. (1) Copper nitrene complexes were computationally characterized, as these complexes have yet to be experimentally isolated. (2) Multireference calculations were carried out on a symmetric C2v chromium dimer derived from the crystal structure of the [(tBu3SiO)Cr(µ-OSitBu3)]2 complex. (3) The T-shaped geometry of a three-coordinate β-diketiminate nickel(I) complex with a CO ligand was compared and contrasted with isoelectronic and isosteric copper(II) complexes. (4) Glutathione synthetase (GS), an enzyme that belongs to the ATP-grasp superfamily, catalyzes the (Mg, ATP)-dependent biosynthesis of glutathione (GSH) from γ-glutamylcysteine and glycine. The free and reactant forms of human GS (wild-type and glycine mutants) were modeled computationally by employing molecular dynamics simulations, as these currently have not been structurally characterized.
Date: May 2007
Creator: Dinescu, Adriana
Partner: UNT Libraries

Modeling wild type and mutant glutathione synthetase.

Description: Glutathione syntethase (GS) is an enzyme that belongs to the ATP-grasp superfamily and catalyzes the second step in the biosynthesis of glutathione. GS has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. Four highly conserved residues were identified in the binding site of human GS. Additionally, the G-loop residues that close the active site during catalysis were found to be conserved. Since these residues are important for catalysis, their function was studied computationally by site-directed mutagenesis. Starting from the reported crystal structure of human GS, different conformations for the wild type and mutants were obtained using molecular dynamics technique. The key interactions between residues and ligands were detected and found to be essential for enzyme activity.
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Date: August 2004
Creator: Dinescu, Adriana
Partner: UNT Libraries

Nucleotide Inhibition of Glyoxalase II

Description: 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 ...
Date: May 1999
Creator: Gillis, Glen S
Partner: UNT Libraries

Synthesis and Study of Glutaryl-S-(ω-aminoalkyl)-L-cysteinylglycines as Inhibitors of Glyoxalase I

Description: 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.
Date: May 1975
Creator: Phillips, Gerald Wayne
Partner: UNT Libraries

Synthesis and Study of Bioactive Compounds: I. Pyrethroids; II. Glutathione Derivatives

Description: Part I: In the first study of pyrethroids, twenty-one novel pyrethroid esters bearing strong electron-withdrawing groups (e.g., halomethylketo and nitro groups) in the double bond side chain of the cyclopropane acid moiety have been synthesized and evaluated for insect toxicity. Rather than the usually employed Wittig reaction for these syntheses, the novel pyrethroid acid moieties were prepared by amino acidcatalyzed Knoevenagel condensations under mild conditions. In the second study of pyrethroids, fourteen pyrethroid-like carbonates were synthesized by condensation of a variety of alcohols and the chloroformates of the corresponding known pyrethroid alcohols.
Date: May 1995
Creator: Chyan, Ming-Kuan
Partner: UNT Libraries

The Mechanisms of Human Glutathione Synthetase and Related Non-Enyzmatic Catalysis

Description: 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.
Date: May 2015
Creator: Ingle, Brandall L.
Partner: UNT Libraries