Kinetic characterization of wild‐type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms

Brandstädter, Christina; Fritz‐Wolf, Karin; Weder, Stine; Fischer, Marina; Hecker, Beate; Rahlfs, Stefan; Becker, Katja

doi:10.1111/febs.14357

Cited by 21 publications

(11 citation statements)

References 76 publications

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“…As a corollary, it can be concluded that the slope observed at the reactivating segment does not correspond to an authentic steady-state velocity. This point must be stressed, because, in some works dealing with the atypical kinetic behavior of TGR [ 11 , 16 , 37 ], the maximal slope in a full time progress curve of GSSG reductase activity has been mistakenly noted as a true steady-state velocity.…”

Section: Discussionmentioning

confidence: 99%

“…A comment concerning a recently published paper dealing with recombinant human TGR [ 11 ]. In such work, the effect of high concentrations of GSSG on enzyme activity was tested.…”

Section: Discussionmentioning

confidence: 99%

“…The presence of the enzyme has been demonstrated in animals [ 1 , 4 ], human [ 11 ], as well as in the parasitic representatives of the flatworms [ 12 – 17 ]. The latter organisms lack the typical glutathione reductase (GR) and TR enzymes, and TGR is the only disulfide reductase involved in the regeneration of the reduced state of both thioredoxin (trx) and glutathione.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, the GSSG-dependent atypical kinetics appear to be a common feature in TGR. Even in the recombinant enzyme from human such atypical kinetic behavior was observed [ 11 ]. However, no detailed molecular mechanism for such phenomenon is yet available.…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, the potential role that the substrate NADPH could play in the atypical kinetics of TGR has not been investigated. In all the works reporting the apparent hysteretic behavior of the enzyme, NADPH concentrations of 100 μ M or higher have been used [ 5 , 7 , 11 , 14 – 17 ]. In order to elucidate the molecular basis of the hysteretic behavior of the enzyme, in the present work, the kinetic mechanism of wild type TGR from T. crassiceps was investigated.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 99%

“…A comment concerning a recently published paper dealing with recombinant human TGR [ 11 ]. In such work, the effect of high concentrations of GSSG on enzyme activity was tested.…”

Section: Discussionmentioning

confidence: 99%