On the reactivity and ionization of the active site cysteine residues of Escherichia coli thioredoxin.

Within various proteins of the thioredoxin family, the stability of the disulfide bond formed reversibly between the two active site cysteine residues, one accessible and one buried, varies widely and is directly correlated with the pKa value of the accessible cysteine thiol group. If applicable to thioredoxin, its stable disulfide bond would imply that its accessible thiol group should have a high pKa value, whereas it has long been considered to be about 6.7, largely on the basis of the pH dependence of its reactivity. Such kinetic data are shown to be inconsistent with known pKa values in this case; the rate constants may reflect effects in the transition state for the reaction, which is catalyzed by thioredoxin, rather than the protein itself. Ionization of the thioredoxin thiol groups was measured indirectly by the pH dependence of the equilibrium constant for their reaction with glutathione and directly by detection of the thiolate anion by its UV absorbance. Both observations indicated that both cysteine thiol groups of thioredoxin ionize with apparent pKa values in the region of 9-10 and that their ionization is not linked strongly to that of any other groups. This conclusion is not incompatible with the other data available and would make thioredoxin consistent with the relationship between thiol group ionization and disulfide stability observed in other members of the thioredoxin family.