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Structural determinants of the catalytic reactivity of the buried cysteine of Escherichia coli thioredoxin.

The structurally homologous thioredoxins and thioltransferases/glutaredoxins possess a solvent-exposed cysteine sulfur which carries out a nucleophilic attack on the target disulfide as well as a structurally adjacent solvent inaccessible thiol. The mechanistic basis of the essentially exclusive redox reactivity of the thioredoxins in contrast to the thiol-disulfide exchange reactions characteristic of the thioltransferases lies in the relative reactivity of the buried cysteine. A stable analog of the mixed disulfide state of Escherichia coli thioredoxin is used to demonstrate a pK value of 11.1 for the solvent inaccessible Cys 35 thiol. NMR chemical shift pH titration analysis indicates a very low dielectric surrounding the Cys 35 sulfur providing a basis for both the elevated pK and the enhanced apparent nucleophilicity. The buried Asp 26 likely serves as the proton sink for the (de)protonation of Cys 35. Relevance to the reactivity of the mammalian protein isomerases is discussed.