Journal
FEBS LETTERS
Volume 477, Issue 1-2, Pages 21-26Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0014-5793(00)01738-5
Keywords
thiol-disulfide oxidoreductase; thioredoxin; redox potential; pK(a) value; disulfide exchange reaction
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Thioredoxin constitutes the prototype of the thioldisulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively C-13(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thioldisulfide oxidoreductases with reasonable accuracy. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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