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

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

Impact of divalent metal cations on the catalysis of peptide bonds: a DFT study

<div><p>Within the ATP-grasp family of enzymes, divalent alkaline earth metals are proposed to chelate terminal ATP phosphates and facilitate the formation of peptide bonds. Density functional theory methods are used to explore the impact of metal ions on peptide bond formation, providing an insight into experimental metal substitution studies. Calculations show that alkaline earth and transition metal cations coordinate with an acylphosphate reactant and aid in the separation of the phosphate leaving group. The critical biochemical reaction is proposed to proceed through the formation of a six-membered transition state in the relatively nonpolar active site of human glutathione synthetase, an ATP-grasp enzyme. While the identity of the metal ion has a moderate impact on the thermodynamics of peptide bond formation, kinetic differences are much sharper. Simulations indicate that several transition metal ions, most notably Cu²⁺, may be particularly advantageous for catalysis. The detailed mechanistic study serves to elucidate the vital role of coordination chemistry in the formation of peptide bonds.</p></div

Informing the Human Plasma Protein Binding of Environmental Chemicals by Machine Learning in the Pharmaceutical Space: Applicability Domain and Limits of Predictability

The free fraction of a xenobiotic in plasma (<i>F</i>_ub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data are scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict <i>F</i>_ub for environmentally relevant chemicals via machine learning techniques. Quantitative structure–activity relationship (QSAR) models were constructed with <i>k</i> nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10–15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99–82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10–0.18<i>F</i>_ub. The models performed best for highly bound chemicals (MAE 0.07–0.12), neutrals (MAE 0.11–0.14), and acids (MAE 0.14–0.17). A consensus model had the highest accuracy across both pharmaceuticals (MAE 0.151–0.155) and environmentally relevant chemicals (MAE 0.110–0.131). The inclusion of the majority of the ToxCast test sets within the AD of the consensus model, coupled with high prediction accuracy for these chemicals, indicates the model provides a QSAR for <i>F</i>_ub that is broadly applicable to both pharmaceuticals and environmentally relevant chemicals

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase <i>in vitro</i>; a clue to the mechanism of 5-oxoprolinuric acidosis?

1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro.2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation.3. Co-incubation of 0.77 μM of GS with NAPQI (25-400 μM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible.4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis

Rapid Prototyping of Physiologically-Based Toxicokinetic (PBTK) Models

Presented at the Annual Society of Toxicology meetin

High Throughput Determinations of Critical Dosing Parameters

Presented at IVIVE Worksho