Dm. Lemaster, STRUCTURAL DETERMINANTS OF THE CATALYTIC REACTIVITY OF THE BURIED CYSTEINE OF ESCHERICHIA-COLI THIOREDOXIN, Biochemistry, 35(47), 1996, pp. 14876-14881
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.