Mechanism of Thioredoxin-Catalyzed Disulfide Reduction. Activation of the Buried Thiol and Role of the Variable Active-Site Residues

Abstract

Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide reduction involves two consecutive thiol-disulfide exchange reactions, that is, nucleophilic substitutions at sulfur (S(N)2@S): first, by one Trx cysteine-thiolate group (Cys-32) at a sulfur atom of the disulfide substrate and, second, by the other Trx cysteine-thiolate group (the buried thiol of Cys-35) at the sulfur atom of the first Trx cysteine. We have investigated the intrinsic nature of such S(N)2@S substitution using the simple CH3S(-) + CH3SSCH3 model and how it is affected by solvation in aqueous solution. Next, we have examined how the behavior of the elementary S(N)2@S steps changes in the more realistic enzyme-substrate model CGPC + CH3SSCH3, which contains the active-site of Trx. In all model reactions, solvation turns the hypervalent trisulfide anion (i.e., the S(N)2@S transition species) from a stable complex into a transition state. Importantly, our analyses suggest that the deprotonation of the buried thiol (which is required before the latter can enter into the second S(N)2@S step) is done by the leaving group evolving from the first S(N)2@S step. Finally, molecular dynamics (MD) simulations, in the gas phase and in water, of CGPC, CGGC, and the corresponding wild-type Trx and P34G Trx show that the activity of the thioredoxin active-site motif (CXYC) is determined not only by the structural rigidity associated with the particular variable residues (XY) but also by the number of amide N-H groups. The latter are involved in the stabilization of the Cys-32 thiolate and thus affect the acidity and nucleophilicity of this residue.

Structural and Mechanistic Insights into Unusual Thiol Disulfide Oxidoreductase

“…These data support the hypothesis that these residues are not involved in the properties of the dithiol active center (33). A second hypothesis involves the thiolate leaving group, which establishes S Ϫ …H-S hydrogen bonds with the thiol group of the buried cysteines (12). However, the mutation of the second cysteine of the substrate shows a dissociation of the intermolecular complex (13).…”

“…For deprotonation of the C-terminal thiol, one hypothesis involves a conserved aspartate residue (11). Recently, it was suggested that when the N-terminal thiolate of Trx attacks its substrate disulfide to form a mixed disulfide complex, the leaving thiol group deprotonates the thiol of the C-terminal active site of Trx (12). Finally another hypothesis proposes that the cysteine is activated for its nucleophilic attack by hydrogen bonds between this residue and the backbone amide of the active site tryptophan (13).…”

Mechanistic insights on the reduction of glutathione disulfide by protein disulfide isomerase

“…From these aspects, the effect of the hydrogen bond donors in the Cys-X-Y-Cys motif seems to find greater acceptance in academia (39)(40)(41)(42). Earlier theoretical studies using cluster models (43)(44)(45) suggested that thiol-disulfide exchange is an S N 2 reaction, in which a nucleophilic thiolate (S nuc ) linearly attacked a disulfide with concomitant dissociation of the latter. In this reaction, the S nuc and the leaving sulfur (S lg ) are approximately parallel to each other and orthogonal to the central sulfur (S ox ), in such a way that the reactant and product disulfides exhibit a torsion of ∼90°around the S-S bond axis (37,(46)(47)(48).…”

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

“…From the point of view of the catalytical effects, the relevant issue is presence of two vicinal cysteines in active site [38][39][40] (Fig. 1).…”