Mf. Jeng et al., PROTON SHARING BETWEEN CYSTEINE THIOLS IN ESCHERICHIA-COLI THIOREDOXIN - IMPLICATIONS FOR THE MECHANISM OF PROTEIN DISULFIDE REDUCTION, Biochemistry, 34(32), 1995, pp. 10101-10105
Proton sharing between acidic groups has been observed in the active s
ires of several enzymes, including bacteriorhodopsin, aspartic proteas
es, and ribonuclease HI. We here report NMR observations suggestive of
proton sharing between cysteine thiols in the active site of the oxid
ation-reduction enzyme thioredoxin. The pK(a)s of the two cysteine thi
ols in the Escherichia coli protein are removed from the expected valu
e of 8.4 by similar to 1. pH unit in either direction, upward and down
ward. Further, the C beta resonances of both residues show clearly the
effects of both of these pK(a)s, indicating that the titrations of th
e two thiol groups are intimately linked. This behavior strongly sugge
sts that the low pK(a) ascribed to the deprotonation of the Cys 32 thi
ol most likely arises through the interaction and close approach of th
e thiol of Cys 35, with the thiolate anion of Cys 32 stabilized throug
h the sharing of the remaining thiol proton, nominally attached to Cys
35. These observations provide a rationale for the mediation of activ
e site pH control, an important aspect of the mechanism of thioredoxin
and other proteins with catalytic thioredoxin domains, such as protei
n disulfide isomerases.