Determination of the ΔpKa between the active site cysteines of thioredoxin and DsbA

Thioredoxin superfamily members share a considerable degree of structural similarity, with a conserved CX i X j C motif at the active site, where C stand for two cysteines that alternate between a reduced thiol and oxidized disulfide states, and X i and X j are two amino acids different in each family member. Despite these similarities, they display very different redox potentials and pKas for the active site dithiol, and fulfill different physiological roles. Thioredoxin, for example, promotes the reduction of disulfide bonds, while DsbA promotes their oxidation in prokaryotic cells. The factors that promote these differences are still not fully understood. However, it is generally accepted that the different stabilities of the redox active disulfide bond depends on the degree of stabilization, in the reduced state, of the thiolate of one of the active site cysteines (nucleophilic cysteine). In this work we have used QM/MM methods to compare and characterize the active site dithiols of both enzymes, and to shed some light on the structural features responsible for the large differences in pKa and redox potential between two homologous enzymes, thioredoxin and DsbA. We have also analyzed the main factors pointed out in the literature as responsible for their different properties. We obtained the value of 4.5 for pKa difference (ΔpKa) between the nucleophilic cysteines of both enzymes, which is in excellent agreement with most of the experimental values. Additionally, we found that the principal differentiating factor responsible for this observed ΔpKa are the α2‐alpha helices, which greatly contribute to the mentioned value, by stabilizing the DsbA thiolate in a much greater extend than the thioredoxin thiolate. A double mutation of the conserved residues Asp26 and Lys57, in thioredoxin, and Glu24 Lys58, in DsbA, by alanines did not change the ΔpKa value; this supports the hypothesis that these residues are not involved in the differentiation of the properties of the active centre dithiol. However, we found out that these residues are important for the stabilization of the nucleophilic thiolate. The X i and X j residues also do not seem to promote the stabilization of the thiolates. In fact, the corresponding double alanine mutants are more stable than the wild‐type enzymes. However, these residues are involved in the differentiation between thioredoxin and DsbA, stabilizing the DsbA thiolate by a larger extent than the thioredoxin thiolate. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 966–975, 2006