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

Abstract

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.

Mechanistic characterization of the thioredoxin system in the removal of hydrogen peroxide

“…The major pathways responsible for H 2 O 2 detoxification in various cell types include the GSH and Trx systems. Mechanistic mathematical models have been recently developed for the GSH system that provides novel quantitative insights into the catalytic mechanisms of GR and GPx enzymes [50, 51] in the removal of H 2 O 2 . However, the same level of mechanistic characterization of the Trx system is not available despite several experimental studies performed on the system (Prx and TrxR enzymes).…”

“…Although experimental data are not available on the effects of products on the TrxR activity with Trx as the substrate, interestingly the developed model, which considers the thermodynamics of the reaction, is able to predict product inhibition of the reaction. Although the rate constants related to the products in our model were not accurately identifiable, the structural and thermodynamics of the TrxR reaction indicate that there is a significant effect of products on the reaction kinetics, similar to the kinetics of GR enzyme [50]. Also experiments performed with the rat TrxR1 enzyme with other model substrates, such as DTNB (5,5′-dithiobis-2-nitrobenzoic acid), suggested that the product NADP + showed mixed type inhibition with respect to NADPH and DTNB [54], validating our model predictions.…”

Altered antioxidant enzyme activity with severity and comorbidities of chronic obstructive pulmonary disease (COPD) in South Indian population

“…Glutathione-s-transferase (GST) is major cytosolic phase II detoxification enzymes which inactivates reactive electrophiles by Glutathione (GSH) dependent mechanism [7,8]. Glutathione reductase (GR) recycles the oxidized glutathione disulfide (GSSG) using NADPH as the reducing co-factor and thereby maintains an appropriate intracellular GSH level in the cell [17].…”

Responses to reductive stress in the cardiovascular system

“…NADPH maintains catalase in an active state [3], and NADPH is necessary to recylce the reducing equivalents used by GPx and Prx enzymes. The enzyme glutathione reductase (Gsr) is an NADPH-dependent enzyme that reduces oxidized glutathione (GSSG) to its reduced form (GSH) thereby providing canonical GPx enzymes with the GSH cofactor necessary to reduce hydrogen and lipid peroxides [4, 5]. Similarly, the redox-active thioredoxin (Trx) proteins are electron donors for some of the Prx enzymes [6], and oxidized Trxs are reduced by NADPH-dependent thioredoxin reductases (Txnrd).…”