STRUCTURAL DETERMINANTS OF THE CATALYTIC REACTIVITY OF THE BURIED CYSTEINE OF ESCHERICHIA-COLI THIOREDOXIN

The structurally homologous thioredoxins and thioltransferases/glutare doxins possess a solvent-exposed cysteine sulfur which carries out a n ucleophilic attack on the target disulfide as well as a structurally a djacent solvent inaccessible thiol. The mechanistic basis of the essen tially exclusive redox reactivity of the thioredoxins in contrast to t he thiol-disulfide exchange reactions characteristic of the thioltrans ferases lies in the relative reactivity of the buried cysteine. A stab le analog of the mixed disulfide state of Escherichia coli thioredoxin is used to demonstrate a pK value of 11.1 for the solvent inaccessibl e Cys 35 thiol. NMR chemical shift pH titration analysis indicates a v ery low dielectric surrounding the Cys 35 sulfur providing a basis for both the elevated pK and the enhanced apparent nucleophilicity. The b uried Asp 26 likely serves as the proton sink for the (de)protonation of Cys 35. Relevance to the reactivity of the mammalian protein isomer ases is discussed.