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.
Highlights
Thioredoxin-glutathione reductase (TGR E.C. 1.8.1.B1) represents an interesting splicing variant of the animal thioredoxin reductase (TR), featured by the presence of a glutaredoxinlike domain appended at the N-terminal end of the Thioredoxin reductase thioredoxin-glutathione reductase (TGR) (TrxR) module [1, 2]
The enzyme retains the homodimeric nature which is common to this set of oxidoreductases, it is outstanding in tertiary structure, total number of redox centers, and wide substrate specificity
In TGR a Grxlike domain has been appended to the N-terminal end of the animal TR module, conferring to the enzyme additional catalytic abilities, notably the reduction of Disulfide form of glutathione DTNB (GSSG) at significant rates, as well as catalysis of thiol-disulfide exchange reactions, including deglutathionylation of mixed disulfides proteinglutathione [1, 4, 6]
Summary
Thioredoxin-glutathione reductase (TGR E.C. 1.8.1.B1) represents an interesting splicing variant of the animal thioredoxin reductase (TR), featured by the presence of a glutaredoxinlike domain appended at the N-terminal end of the TrxR module [1, 2]. Unlike typical TR, TGR is able to reduce oxidized glutathione (GSSG) at significant rates and to perform thiol/disulfide exchanges [1, 4], making it a multifunctional enzyme. Such additional catalytic capabilities are dependent on the presence of the Grx-like domain. Both the Grx-like domain and the C-terminal redox center of the enzyme are essential in the reduction of GSSG, as derived from site-directed mutagenesis studies [4,5,6,7]. It has been proposed that, during the catalytic cycle of TGR with GSSG as the substrate, electrons must be shuttled from the reduced selenol/thiol couple toward the redox center of the Grx-like domain [9, 10], involving a large conformational transition of the C-terminal arm of the neighbor subunit
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