INFLUENCE OF ACIDIC RESIDUES AND THE KINK IN THE ACTIVE-SITE HELIX ONTHE PROPERTIES OF THE DISULFIDE OXIDOREDUCTASE-DSBA

The catalytic disulfide bond Cys(30)-Cys(33) of the disulfide oxidored uctase 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'(0) = -125 mV) mainly results from the low pK(a) of 3.4 of its Cys(30) thiol. To investigate the role of the kink and the electrostatic contribution of Glu(37) an d Glu(38) to the redox properties of DsbA, we have characterized a ser ies of DsbA variants (Delta 38-40, Delta 38-40/H41P, E37Q, E38Q, and E 37Q/E38Q). In contrast to theoretical predictions, the redox potential s of the variants are almost unchanged, and the pK(a) values of Cys(30 ) do not differ by more than 0.5 units from that of DsbA wild type. Al l variants show the same in vivo activity and dependence of redox pote ntial on ionic strength as the wild type. The mutations have no influe nce on the polypeptide specificity of the protein, which is independen t of the isoelectric point of the polypeptide substrate and most prono unced at acidic pH. We conclude that neither the kink. in the active-s ite helix nor Glu(37) and Glu(38) are critical for the physical proper ties of DsbA.