A study of the kinetic mechanism followed by glutathione reductase from mycelium of Phycomyces blakesleeanus

Montero, Sol; Arriaga, Dolores de; Busto, Félix; Soler, Joaquı́n

doi:10.1016/0003-9861(90)90230-v

Cited by 10 publications

(48 citation statements)

References 21 publications

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“…The observed data both in the presence and absence of products were consistent with a branched mechanism with GSSG-dependent substrate-inhibition by NADPH. The study also hypothesized that, at low concentrations of GSSG, the ping-pong mechanism prevails, whereas at high concentrations of GSSG, the sequential ordered mechanism appeared to dominate [30]. In this work, Montero et al also concluded that the enzyme activity is inhibited at acidic pH as the GSSG concentration is increased.…”

Section: Introductionmentioning

confidence: 60%

“…However, the effects of products were not reported. The initial-velocity data of Montero et al [30] from P. blakesleeanus was the first study that extensively characterized the kinetic mechanism by performing experiments on forward reaction and effects of both products and pH on the initial-velocities. The observed data both in the presence and absence of products were consistent with a branched mechanism with GSSG-dependent substrate-inhibition by NADPH.…”

Section: Introductionmentioning

confidence: 99%

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A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

Pannala

Bazil

Camara

et al. 2013

Free Radical Biology and Medicine

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Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2·−) and hydroxyl radicals (OH·) non-enzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated with the catalytic action of GR. Though GR kinetic mechanism has been extensively studied under different experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of the current study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observations that GR operates via both ping-pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate-inhibition of GR activity by GSSG and bi-modal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP+ with ping-pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS scavenging system to understand protection of cells under normal and oxidative stress conditions.

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Section: Introductionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

Pannala

Bazil

Camara

et al. 2013

Free Radical Biology and Medicine

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“…Maintaining an adequate level of reduced cellular GSH over GSSG is especially important for cells to fight against oxidative stresses and xenobiotics 35, 36. The kinetics and mechanism of the glutathione reductase-catalyzed reactions have been studied 33, 34, 37, 38. The most widely used tool to analyze the kinetics and mechanism of glutathione reductase is UV/Vis absorbance as described by Racker,39 which relies on the decrease in the absorbance at 340 nm of NADPH.…”

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confidence: 99%

Integration of a Precolumn Fluorogenic Reaction, Separation, and Detection of Reduced Glutathione

Ferrance

Landers

et al. 2010

Anal. Chem.

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Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually carry out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 × 104 M−1s−1. The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of β-NADPH (β-Nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half maximum (FWHM) for the internal standard were 2.5%, 2.0%, 1.0%, respectively with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and ~10−18 mol, respectively. The Michaelis constants (Km) for GSSG and β-NADPH were found to be 40 ± 11 μM and 4.4 ± 0.6 μM, respectively, comparable with those obtained from UV/Vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation and detection for continuous GSH determinations.

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“…The inhibition by NADP + was competitive with respect to NADPH. In Phycomyces blakesleeanus GR, Ki NADP + was calculated as 61 ± 4.2 μM [26].…”

Section: Product Inhibition Of Bovine Kidney Cortex Glutathione Reducmentioning

confidence: 99%

“…It has been established that electrons flow from NADPH to the substrate oxidized glutathione (GSSG) via flavin and the redoxactive protein disulfide bridge [3]. Detailed steady-state kinetic analysis indicated that the reaction of GR acted according to both ping-pong and sequential mechanisms depending on substrate concentrations [4,5]. The function of GR is to reduce the GSSG by using NADPH as the source of reducing equivalents [6].…”

Section: Introductionmentioning

confidence: 99%

Purification and Kinetics of Bovine Kidney Cortex Glutathione Reductase

Tandogan

Ulusu²

2010

PPL

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A study of the kinetic mechanism followed by glutathione reductase from mycelium of Phycomyces blakesleeanus

Cited by 10 publications

References 21 publications

A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

Integration of a Precolumn Fluorogenic Reaction, Separation, and Detection of Reduced Glutathione

Purification and Kinetics of Bovine Kidney Cortex Glutathione Reductase

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