Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present in the reduced form of thioredoxin from Escherichia coli.
Only one of the sulfhydryl groups from Cys-32 and Cys-35 in the active center of native Escherichia coli thioredoxin-(SH)2 was alkylated by excess iodoacetic acid at pH values below 8.0. Both groups reacted in the protein denatured with 4.5 M guanidine hydrochloride. The second order rate of alkylation of thioredoxin-(SH)2 with 1 eq of iodoacetic acid was pH-dependent and showed independent initial reactions of one thiolate ion with a pK value of 6.7 and a second with a pK value close to 9.0. The same pH dependence was observed for alkylation with iodoacetamide but the apparent rate constant, 107 M-1 S-1 at pH 7.2, was about 20-fold higher than the corresponding rate with iodoacetate. The sulfhydryl group with a pK value of 6.7 was shown to belong to Cys-32 by labeling thioredoxin with [14C]iodoacetic acid followed by complete alkylation with [3H]iodoacetate and amino acid sequence analysis of peptides from the active center. The abnormally low pK value of Cys-32 is suggested to arise by electrostatic influence from a positive charge on the amino group of Lys-36. A mechanism of action for thioredoxin-(SH)2 as a protein disulfide reductase has been formulated. This is based on an initial nucleophilic attack by the thiolate of Cys-32 with the formation of an unstable transient mixed disulfide involving Cys-32 and one of the sulfurs in the substrate. This is followed by a conformational change and a nucleophilic attack of Cys-35 to give the 14-membered disulfide ring in thioredoxin-S2 and the dithiol of the substrate.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates; D007700:Kinetics; D011485:Protein Binding; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D007461:Iodoacetates; D013879:Thioredoxins; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine