Role of the charged groups of glutathione disulfide in the catalysis of glutathione reductase: crystallographic and kinetic studies with synthetic analogs

Abstract

Six analogues of glutathione disulfide were synthesized. All of them involved the abolishment of charges, either by amidation of carboxylates or by removal of amino groups. Four of these analogues could be bound to crystalline oxidized glutathione reductase, and their binding modes could be established by X-ray analyses at 2.4-A resolution. All six analogues were catalytically processed; the kinetic parameters were determined. The two analogues that did not bind in the crystals had by far the poorest catalytic efficiencies. Kinetic parameters together with X-ray data show the influence of each charged group on binding and catalytic rate. Data analysis indicates that the enzyme avoids processing of incorrect substrates in two ways: First, it reduces their binding strengths and/or enforces displacement of catalytically important substrate parts. Furthermore, it forms a fragile cluster of bound substrate and catalytically competent residues, which is unbalanced by incorrect parts of the substrate such that catalysis is prevented. A scouting microcalorimetric study using glutathione disulfide yielded a binding enthalpy of -103 (+/- 10) kJ/mol at 25 degrees C and a heat capacity change of -8 (+/- 1) kJ.mol-1.K-1. The study showed that it is feasible to measure these parameters as a function of substrate modification.

Characterization of Glutathione Amide Reductase from Chromatium gracile

“…NADPH oxidation was monitored as the decrease in A 340 in a 0.5-ml reaction mixture containing 100 mM potassium phosphate buffer, pH 7.9, 0.1 mM EDTA, 0.4 units GR, 500 M GSH, 150 M NADPH, 50 g/ml partially purified Prx/Grx, and 100 M hydroperoxide. a Values were taken from the work of Janes and Schulz (58).…”

“…b Calculated values derived from a V max value for the human GR enzyme of 220 units/mg (58). The subunit mass of human GR was calculated from the amino acid sequence (43).…”

Substrate interactions between trypanothione reductase and N1‐glutathionylspermidine disulphide at 0.28‐nm resolution

“…Schulz and co-workers have dissected, in considerable detail, the structurelactivity relationships of human glutathione reductase (Janes and Schulz, 1991;Schulz 1987, 1989). The glutathione re- ductase structure has subsequently been utilised by a number of groups as the template onto which a model of trypanothione reductase has been constructed using computer graphics methods Smith et al, 1991 ;Horjales et al, 1993).…”

Use of transferred nuclear‐Overhauser‐effect spectroscopy to measure the bound conformation of a disulphide‐replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase

“…An obvious first step in designing an enzyme inhibitor is to make a substrate analogue. We now report the synthesis and properties of the L-cystathionine analogue (3) of glutathione disulphide (2), in which the scissile -S-S-bond of the latter has been replaced by the sulphide system -CH,-S-. In this paper we report the enzymology of (3) with yeast glutathione reductase as well as NMR spectroscopic studies of its binding to this enzyme.…”