Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

Abstract

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

Thioredoxin-dependent system. Application of inhibitors

“…TrxR2 contains a domain composed of 33 amino acids situated at the N-terminal portion of the enzyme, which is responsible for mitochondrial translocation of enzyme molecules. TrxR3 can reduce both Trx and glutathione (GSH) and is therefore referred to as thioredoxin glutathionereductase (TGR) [17][18][19][20] . It contains a TGR structure at the N-terminal motif which contains an additional glutaredoxin domain so that the enzyme can act as a thioredoxin reductase, glutathione reductase, and glutaredoxin, the purpose of which is to reduce mixed disulphides in proteins produced by glutathionylation, among other processes 21 .…”

Descriptive Analysis of Transient-State Observations for Thioredoxin/Glutathione Reductase (Sec597Cys) from Schistosoma mansoni

“…TGR enzymes have been studied using X-ray crystallography, redox titrations, and steady-state analysis. ,− We report the first comprehensive transient-state analysis of a TGR enzyme. Changes in the FAD cofactor absorption spectrum indicate rapid fractional reduction by NADPH and rapid reoxidation to ultimately form a quasi-equilibrium distribution of electrons among all redox active entities within the enzyme.…”

Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy

“…Hysteresis may be caused by substrate, solvent, pH, or applied voltage − (Table S1). − In addition, hysteretic enzymes play an important role in the regulation of metabolic pathways under particular physiological conditions ,,,, coupling enzymatic activity to the oscillation of a metabolite concentration. − For instance, the loss of the cooperativity of the monomeric human glucokinase (GCK) impacts glucose homeostasis . GCK plays a crucial role in glucose metabolism in the pancreas and liver by catalyzing the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, a pivotal step in glucose metabolism.…”