Influence of acidic residues and the kink in the active-site helix on the properties of the disulfide oxidoreductase DsbA.

The Journal of Biological Chemistry
J HenneckeR Glockshuber

Abstract

The catalytic disulfide bond Cys30-Cys33 of the disulfide oxidoreductase DsbA from Escherichia coli is located at the amino terminus of an alpha-helix, which has a kink caused by insertion of a tripeptide (residues 38-40). The oxidative force of DsbA (E'O = -125 mV) mainly results from the low pKa of 3.4 of its Cys30 thiol. To investigate the role of the kink and the electrostatic contribution of Glu37 and Glu38 to the redox properties of DsbA, we have characterized a series of DsbA variants (delta38-40, delta38-40/H41P, E37Q, E38Q, and E37Q/E38Q). In contrast to theoretical predictions, the redox potentials of the variants are almost unchanged, and the pKa values of Cys30 do not differ by more than 0.5 units from that of DsbA wild type. All variants show the same in vivo activity and dependence of redox potential on ionic strength as the wild type. The mutations have no influence on the polypeptide specificity of the protein, which is independent of the isoelectric point of the polypeptide substrate and most pronounced at acidic pH. We conclude that neither the kink in the active-site helix nor Glu37 and Glu38 are critical for the physical properties of DsbA.

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