Abstract
Disulfide oxidoreductases are structurally related proteins that share the thioredoxin fold and a catalytic disulfide bond that is located at the N terminus of an alpha-helix. The different redox potentials of these enzymes varying from -270 mV for thioredoxin to -125 mV for DsbA have been attributed to the lowered pKa values of their nucleophilic, active-site cysteines and the difference in thermodynamic stability between their oxidized and reduced forms (DeltaDeltaGox/red). The lowered pKa of the nucleophilic cysteine thiols was proposed to result from favorable interactions with the helix dipole and charged residues in their vicinity. In this study, we have eliminated all charged residues in the neighborhood of the active-site disulfide of DsbA from Escherichia coli to analyze their contribution to the physicochemical properties of the protein. We show that the conserved charge network among residues Glu24, Glu37, and Lys58 stabilizes the oxidized form of DsbA and thus does not cause the high redox potential of the enzyme. The pKa values of the nucleophilic cysteine (Cys30) and the redox potentials of the DsbA variants E24Q, E37Q, K58M, E24Q/K58M, E37Q/K58M, E24Q/E37Q, E24Q/E37Q/K58M, and E24Q/E37Q/E38Q/K58M are similar to those of DsbA wild type. The redox potentials of the variants neither correlate with the Cys30 pKa values nor with the DeltaDeltaGox/red values, demonstrating that the relationship between these parameters is far more complex than previously thought.
Highlights
The formation of disulfide bridges is essential for folding and stability of many secretory proteins
The disulfide oxidoreductase DsbA is mainly responsible for the efficient formation of disulfide bonds in proteins that are exported to the periplasm of Escherichia coli [15, 16]
The residues of the XX dipeptide have been shown to influence the pKa of DsbA Cys30 thiol [19], it is still unclear whether charged residues in the vicinity of the activesite helix or the helix dipole alone are responsible for the low pKa of Cys30 in reduced wild type DsbA
Summary
Alexander Jacobi‡, Martina Huber-Wunderlich, Jens Hennecke, and Rudi Glockshuber. From the Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule-Honggerberg, CH-8093 Zurich, Switzerland. The structure of oxidized DsbA provides no hints concerning its unusual physicochemical properties These are reflected by the extremely low pKa of the nucleophilic active-site cysteine, Cys, which has a value of about 3.5 (18 –20). Besides charged residues in the vicinity of the active-site helix, other explanations have been proposed for a lowered pKa of the nucleophilic thiol in human and E. coli thioredoxin such as the dipole of the ␣-helix (29 –31), hydrogen bonding between the Cys thiolate and the amide of Cys35 [30, 32, 33], and a shared proton between the two sulfurs of Cys and Cys35 [26, 34]. To further analyze the function of Glu in the context of the neighboring residues Glu and Lys, we investigated the properties of the double variant E37Q/K58M, the triple variant E24Q/E37Q/K58M, and the quadruple variant E24Q/E37Q/ E38Q/K58M, which lacks all negatively charged residues less than 13 Å apart from the sulfur of Cys
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