Hysteretic Behaviour and Gssg Substrate Inhibition Shown by Glutathione Reductase FromPhycomyces Blakesleeanus

Abstract

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.

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

“…Similarly, Figure 6B shows the effect of pH on GR activity that was simulated for four different [GSSG] (0.25, 0.5, 1 and 5 mM) with varying pH at a fixed [NADPH] (0.1 mM). The model observed initial-velocity of GR was inhibited at both acidic and alkaline pH values and inhibitory pattern depends on [GSSG] as observed experimentally [30, 34, 46]. …”

“…Using the proposed unified mechanistic scheme in our study, where random protonation and GSSG binding of intermediate complexes E-NADPH to form single protonated active intermediate enzyme complexes and formation of the diprotonated dead-end complexes in the sequential branch was included, the model is able to describe such pH-dependent data. It appears that at [GSSG] above 0.1 mM, the flux through the sequential branch increases and the high concentration of protons leads to the accumulation of the dead-end proton complexes [46]. Although De Arriaga et al [46] suggested the involvement of cooperativity in proton binding depending on the GSSG and pH value, we assumed a unity cooperativity coefficient for proton binding for simplification.…”

“…It appears that at [GSSG] above 0.1 mM, the flux through the sequential branch increases and the high concentration of protons leads to the accumulation of the dead-end proton complexes [46]. Although De Arriaga et al [46] suggested the involvement of cooperativity in proton binding depending on the GSSG and pH value, we assumed a unity cooperativity coefficient for proton binding for simplification. The model simulation studies strengthen the notion that, under acidic conditions (e.g.…”

“…This proton-bound ternary complex subsequently leads to the formation of reaction products while releasing the free enzyme E . The random protonation of NADPH-bound intermediate complex and proton-bound dead-end complexes in the sequential branch were motivated by the experimentally observed bi-modal behavior of GR activity with pH [34] and GSSG concentration dependent accumulation of reaction products at acidic pH [30, 46]. …”