ON THE REACTIVITY AND IONIZATION OF THE ACTIVE-SITE CYSTEINE RESIDUESOF ESCHERICHIA-COLI THIOREDOXIN

Within various proteins of the thioredoxin family, the stability of th e disulfide bond formed reversibly between the two active site cystein e residues, one accessible and one buried, varies widely and is direct ly correlated with the pK(a) value of the accessible cystrine thiol gr oup. If applicable to thioredoxin, its stable disulfide bond would imp ly that its accessible thiol group should have a high pK(a) value, whe reas 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 t o be inconsistent with known pK(a) values in this case, the rate const ants may reflect effects in the transition state for the reaction, whi ch is catalyzed by thioredoxin, rather than the protein itself. Ioniza tion of the thioredoxin thiol groups was measured indirectly by the pH dependence of the equilibrium constant for their reaction with glutat hione and directly by detection of the thiolate anion by its UV absorb ance. Both observations indicated that both cysteine thiol groups of t hioredoxin ionize with apparent pK(a) values in the region of 9-10 and that their ionization is not linked strongly to that of any other gro ups. This conclusion is not incompatible with the other data available and would make thioredoxin consistent with the relationship between t hiol group ionization and disulfide stability observed in other member s of the thioredoxin family.