CHARACTERIZATION OF ESCHERICHIA-COLI THIOREDOXIN VARIANTS MIMICKING THE ACTIVE-SITES OF OTHER THIOL DISULFIDE OXIDOREDUCTASES/

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corres ponds to any amino acid). Despite their structural similarities, the e nzymes have very different redox properties, which is reflected by a 1 00,000-fold difference in the equilibrium constant (K-eq) with glutath ione between the most oxidizing member, DsbA, and the most reducing me mber, thioredoxin. Here we present a systematic study on a series of v ariants of thioredoxin from Escherichia call, in which the Xaa-Xaa dip eptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be s tronger oxidants than the wild-type, with the order wild-type < PDI-ty pe < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-l ike variant has a 420-fold decreased value of K-eq, corresponding to a n increase in redox potential by 75 mV. While oxidized wild-type thior edoxin is more stable than the reduced form (Delta Delta G(ox/red) = 1 6.9 kJ/mol), both redox forms have almost the same stability in the va riants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pK(a) valu e of thioredoxin's nucleophilic active-site thiol (Cys32). A pK(a) of 7.1 was measured for Cys32 in the reduced wild-type. All variants show ed a lowered pK(a) of Cys32, with the lowest value of 5.9 for the glut aredoxin-like variant. A correlation of redox potential and the Cys32 pK(a) value could be established on a quantitative level. However, the predicted correlation between the measured Delta Delta G(ox/red) valu es and Cys32 pK(a) values was only qualitative.