Characterization of Escherichia coli thioredoxin variants mimicking the active‐sites of other thiol/disulfide oxidoreductases
Open Access
- 1 May 1998
- journal article
- research article
- Published by Wiley in Protein Science
- Vol. 7 (5) , 1233-1244
Abstract
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 corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000‐fold difference in the equilibrium constant (Keq) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa‐Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild‐type, with the order wild‐type < PDI‐type < DsbA‐type < glutaredoxin‐type. The most oxidizing, glutaredoxin‐like variant has a 420‐fold decreased value of Keq, corresponding to an increase in redox potential by 75 mV. While oxidized wild‐type thioredoxin is more stable than the reduced form (ΔΔGox/red = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH‐dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active‐site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild‐type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin‐like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured ΔΔGox/red values and Cys32 pKa values was only qualitative.Keywords
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