Glutathione synthesis and turnover in the human erythrocyte: alignment of a model based on detailed enzyme kinetics with experimental data

Abstract

The erythrocyte is exposed to reactive oxygen species in the circulation and also to those produced by autoxidation of hemoglobin. Consequently, erythrocytes depend on protection by the antioxidant glutathione. Mathematical models based on realistic kinetic data have provided valuable insights into the regulation of biochemical pathways within the erythrocyte but none have satisfactorily accounted for glutathione metabolism. In the current model, rate equations were derived for the enzyme-catalyzed reactions, and for each equation the nonlinear algebraic relationship between the steady-state kinetic parameters and the unitary rate constants was derived. The model also includes the transport processes that supply the amino acid constituents of glutathione and the export of oxidized glutathione. Values of the kinetic parameters for the individual reactions were measured predominately using isolated enzymes under conditions that differed from the intracellular environment. By comparing the experimental and simulated results, the values of the enzyme-kinetic parameters of the model were refined to yield conformity between model simulations and experimental data. Model output accurately represented the steady-state concentrations of metabolites in erythrocytes suspended in plasma and the changing glutathione concentrations in whole and hemolyzed erythrocytes under specific experimental conditions. Analysis indicated that feedback inhibition of gamma-glutamate-cysteine ligase by glutathione had a limited effect on steady-state glutathione concentrations and was not sufficiently potent to return glutathione concentrations to normal levels in erythrocytes exposed to sustained increases in oxidative load.

FIGURE 1.

Scheme of the metabolic reactions involving glutathione in the human erythrocyte. The reaction scheme was the basis of the mathematical model. Glnase, glutaminase; AlaTase, alanine aminotransferase; NACase, NAC deacetylase; GSHSase, glutathione synthetase; γ-GCTase, γ-glutamylcyclotransferase. Membrane transport is via the amino acid transporters for alanine, glutamine, cysteine, glycine, the anion exchange protein for the majority of NAC transport, and the multidrug resistance-associated protein (MPR1) for the primary active export of GSSG.

FIGURE 2.

Representation of the mechanisms of the enzymatic reactions included in the model using Cleland's convention (33, 34). All of the abbreviations are as defined in Fig. 1. Reactions: A, GCL (13); B, glutathione synthetase (72); C, GSSGR (12); D, N-acetylcysteine deacetylase; E, alanine aminotransferase; F, glutaminase; G, γ-glutamylcyclotransferase (73); H, the reaction mechanism assumed for GSSG_i active export. References are included when the mechanism for the particular enzyme has been reported previously. For the remaining enzymes, ordered sequential reactions were assumed.

FIGURE 3.

Extracellular NAC as a substrate for glutathione synthesis. CDNB-depleted erythrocytes were washed three times at room temperature in incubation solution at pH 7.55 with 10 mm glucose, 1.0 mm glutamine and glycine, and varying concentrations of NAC. Erythrocytes were then suspended to give Ht = 10% in the same solution and incubated for 220 min at 37 °C. The TFG concentration was measured in samples taken at 20-min intervals; these values were used to estimate the linear rate of TFG synthesis ± S.E. Data are from one of three experiments, all of which gave similar results. The solid line represents the model output with NAC deacetylase, K_m = 252 μm, and the dashed line represents K_m = 911 μm. Simulated and experimental data are presented as a proportion of the maximum rate.

FIGURE 4.

Inhibition of GCL-catalyzed γ-GluCys production by GSH. Rates of γ-GluCys production are relative to the maximum rate in the absence of GSH. The open symbols denote the calculated values of the initial (0–10 s) rates of γ-GluCys production ±5% error in the presence 0 mm GSH (○) and 10 mm GSH (▵). The lines are best fits of the Michaelis-Menten equation to these data. See Fig. 1A and the “Supplemental Information” for the reaction scheme and steady-state kinetic expression for GCL. The solid symbols denote data from Richman and Meister (Ref. , Figs. 1 and 3 A therein) measured in the absence of GSH (●) and at 10 mm GSH (▲). The simulated data were calculated with the concentrations of ATP, cysteine, and glutamate used by Richman and Meister (9). A, competitive inhibition of glutamate binding by GSH. In these simulations cysteine concentration was set at 2.0 mm and ATP at 1.0 mm. B, inhibition of cysteine incorporation into γ-GluCys by GSH. Glutamate concentration was set at 2.0 mm and ATP at 1.0 mm.

FIGURE 5.

GSH inhibition of TFG synthesis. The synthesis rates are relative to the maximum value. A, erythrocytes were depleted of GSH by incubation in 0.5 mm CDNB and then hemolyzed by sonification. GSH was added to the concentrations indicated, and the rate of TFG appearance was measured over a 35-min incubation period at 37 °C. The symbols denote the mean ± S.E. of the results from three separate experiments. For the hemolysate the rates of change of TFG predicted using the model are represented by the solid line, whereas the dashed line denotes model output when the value of K_iGSH was increased by 10%. When K_iGSH was decreased by 10%, the calculated values fell below but too close to the solid line to be distinguished. B, erythrocytes were exposed to a range of concentrations of CDNB (0.0 to 0.4 mm) that yielded TFG concentrations of 0.2 to 2.3 mm. All symbols and error bars denote mean ± S.E., and the open and closed symbols represent data from two separate experiments. The model calculations of the total rates of production of TFG (i.e., rate of increase of TFG within the erythrocyte plus the rate of export of TFG from the erythrocyte) under the conditions of the experiment fell along the solid line. Calculated values of the rate of change of TFG concentration in the erythrocytes are denoted by the dot-dashed and dashed lines, with the values of the second-order rate constant for GSH oxidation (k_GSHox) at 0.0224 and 0.0467 mol⁻¹ L h⁻¹, respectively.

FIGURE 6.

Modeled attainment of steady-state concentrations of substrates, intermediates, and products of glutathione metabolism in erythrocytes under approximated in vivo conditions. A, concentrations of substrates and intermediates: ●, cysteine; ▵, glutamine; ○, glutamate; □, glycine; ▼, γ-GluCys. B, concentrations of products: ▵, erythrocyte TFG (i.e., [GSH_i] + 2 × [GSSG_i]); □, total synthesized glutathione (i.e., [GSH_i] + 2 × ([GSSG_i] + [GSSG_e])); ○, extracellular TFG (i.e., 2 × [GSSG_e]); (●), 5-oxoproline. The concentrations of extracellular TFG (2 × [GSSG_e]) were expressed in mmol(liter of erythrocyte water)⁻¹ for direct comparison with other values in the figure.

FIGURE 7.

Perturbations of steady-state conditions in erythrocytes under approximated in vivo conditions. The connected data points indicate the results of model calculations of erythrocyte TFG concentrations. A, symbols denote the following: ●, at the steady state; ▵, after an increase; □, after a decrease of the initial intracellular [TFG] by 20%. B, erythrocyte [TFG] values at the steady state are indicated by closed circles (●). The remainder of the symbols denote erythrocyte [TFG] values resulting from a sustained increase in the value of the unitary rate constant for oxidation of GSH (k_GSHox) by 20%: ▲, no other change; (◇, increase in GCL V_max by 50%; ▵, increase in extracellular cysteine concentration by 50%; □, increase in the V_max for glutamine influx by 10%; ○, increase in the V_max of GSSGR by 20%. The slow rate of increase seen in all the time courses was deduced to be due to a slight difference in the rate of TFG synthesis (20.495 μmol(liter of erythrocytes)⁻¹ h⁻¹) and export (20.492 μmol(liter of erythrocytes)⁻¹ h⁻¹) at the quasi steady state. Running the model for a period of 12 simulated days emphasized the effects of this difference.