Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases

Thioredoxin constitutes the prototype of the thioldisulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the cons ensus 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 contra st, the more C-terminal cysteine is buried and only little is known about i ts effective pK(a) during catalysis of disulfide exchange reactions. Here w e have analyzed the pK(a) values of the active-site thiols in wild type thi oredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectrosc opy, using selectively C-13(beta)-Cys-labeled proteins. We find that the ef fective pK(a) of the buried cysteine (pK(b)) of the variant is increased, w hile the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relativ e to the corresponding pK(a) values in the wild type. We propose two empiri cal 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 Elsev ier Science B.V. All rights reserved.