Rate constants and equilibrium constants for thiol-disulfide interchange reactions involving oxidized glutathione

Abstract

The rate of reduction of oxidized glutathione (GSSG) to glutathione (GSH) by thiolate anions (RS-) follows a Brjánsted relation in pXas of the conjugate thiols (RSH): /5nuc a 0.5. This value is similar to that for reduction of Ellman's reagent: jSnue ~0.4-0.5. Analysis of a number of rate and equilibrium data, taken both from this work and from the literature, indicates that rate constants, k, for a range of thiolate-disulfide interchange reactions are correlated well by equations of the form log k = C + |6nucPXanu<: + /3cpAfac + 0igpKjg (nuc = nucleophile, c = central, and Ig = leaving group sulfur): eq [36][37][38] give representative values of the Br^nsted coefficients. The values of these Br^nsted coefficients are not sharply defined by the available experimental data, although eq 36-38 provide useful kinetic models for rates of thiolate-disulfide interchange reactions. The uncertainty in these parameters is such that their detailed mechanistic interpretation is not worthwhile, but their qualitative interpretation-that all three sulfur atoms experience a significant effective negative charge in the transition state, but that the charge is concentrated on the terminal sulfurs-is justified. Equilibrium constants for reduction of GSSG using , -dithiols have been measured. The reducing potential of the dithiol is strongly influenced by the size of the cyclic disulfide formed on its oxidation: the most strongly reducing dithiols are those which can form six-membered cyclic disulfides. Separate equilibrium constants for thiolate anion-disulfide interchange (As~) and for thiol-disulfide interchange (A'SH) have been estimated from literature data: Ks~is roughly proportional to 2 Xa is the difference between the pKas of the two thiols involved in the interchange. The contributions of thiol pA"a values to the observed equilibrium constants for reduction of GSSG with , -dithiols appear to be much smaller than those ascribable to the influence of structure on intramolecular ring formation. These equilibrium and rate constants are helpful in choosing dithiols for use as antioxidants in solutions containing proteins: dithiothreitol (DTT), 1,3-dimercapto-2-propanol (DMP), and 2-mercaptoethanol have especially useful properties.

Mia40 targets cysteines in a hydrophobic environment to direct oxidative protein folding in the mitochondria

“…The Brønsted coefficient for thiol/disulphide exchange reactions is about 0.5 (ref. 27), and therefore a fourfold difference in reactivity would be expected between Cys26 and Cys36. The observed 400-fold difference is thus not explained by the difference in pK.…”

“…Sulphur atoms of the structural disulphides are coloured yellow, cysteines are numbered according to the amino-acid sequence. The first CX 9 C motif is coloured purple (residues [26][27][28][29][30][31][32][33], the second CX 9 C motif (residues 47-57) is magenta and the hydrophobic residues 37-39 following Cys36 are blue. The residues of the MISS/ITS sequence (F50, I51, Y54) and I37, L38, F39 are shown in ball and sticks representation.…”

Protection/Deprotection of Surface Activity and Its Applications in the Controlled Release of Liposomal Contents

“…The residue was purified by column chromatography on silica gel using a mixture of dichloromethane/methanol (15:1 to 10:1) as the eluent to give the product as a white powder (0.55 g, 85%). 1 40,30,20,10, and 0 μL of the above stock solution. The above aqueous solution of pyrene was added to make the total volume of each sample 2 mL.…”

Redox properties of protein disulfide isomerase (dsba) from escherichia coli

“…The commonly used value for the GSH/GSSG couple at pH 7.0 (EA) is -0.24 f 0.01 V. This value has been determined at 40 "C using the NAD+/NADH2 pair as reference and was calculated from a value of = lo3 for the equilibrium constant (Rost & Rapoport, 1964). In this study, we have used the value of -0.205 V for the glutathione couple, which has been determined very accurately at 30 "C using the lipoamide/ NAD+ system (Szajewski & Whitesides, 1980). Because standard redox potentials are temperature dependent, all redox equilibria were measured at this temperature (30°C).…”

“…Assuming a redox potential of about -0.2 V for a reduced, folding polypeptide chain, which is likely to be in the range of monothiol species such as glutathione (Szajewski & Whitesides, 1980), and taking a mean value of -0.1 V for bacterial and eukaryotic PDI, an equilibrium constant Keq in the range of to is obtained according to the Nernst equation. Therefore, the rate constant for the oxidation of the polypeptide chain (k,,) by PDI would be 103-104 times higher than the rate constant for the reduction of a disulfide in the polypeptide (kred).…”