The changes in reactivity of the less reactive SH groups of glyceraldehyde-3-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 220.127.116.11) apoenzyme towards thiol reagents were studied during the course of dissociation into dimers and inactivation of the enzyme induced by stoichiometric amounts of ATP. Neither the reactive SH group of Cys-149 nor that of Cys-153 (the latter being the second cysteinyl group in order of reactivity towards thiol reagents) is ozidized during dissociation and inactivation caused by ATP treatment. However, during this process one SH group per subunit which was previously masked in the native tetramer is oxidized. This cysteinyl residue corresponds to Cys-281 as identified anallytically. The molecular weight of the ATP-treated enzyme is 60 000 even in the presence of 6 M urea. Both enzymatic activity and the oxidized SH group can be fully recovered by treatment with 2-mercaptoethanol. We conclude that during dissociation into dimers by ATP, residues Cys-281 form interchain S-S bridges within the dimers.
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