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Volume 272, Number 1, Issue of January 3, 1997 pp. 189-195
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Influence of Acidic Residues and the Kink in the Active-site Helix on the Properties of the Disulfide Oxidoreductase DsbA

(Received for publication, August 7, 1996, and in revised form, October 7, 1996)

From the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

The catalytic disulfide bond Cys³⁰-Cys³³ of the disulfide oxidoreductase DsbA from Escherichia coli is located at the amino terminus of an -helix, which has a kink caused by insertion of a tripeptide (residues 38-40). The oxidative force of DsbA (E₀ = 125 mV) mainly results from the low pK_a 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 (38-40, 38-40/H41P, E37Q, E38Q, and E37Q/E38Q). In contrast to theoretical predictions, the redox potentials of the variants are almost unchanged, and the pK_a 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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