Identification of different classes of nonessential sulfhydryl groups in Escherichia coli adenylosuccinate synthetase

Reaction of Escherichia coli adenylosuccinate synthetase with the thiol reagent 5,5′-dithiobis(2-nitro-benzoic acid) (DTNB) or N-ethylmaleimide (NEM) leads to modification of one cysteine residue per enzyme monomer without significant loss of enzyme activity. Modification of a second cysteine residue occurs under mild denaturing conditions (3.5 m urea), and derivatization of this thiol followed by dialysis results in complete loss of enzyme activity. The remaining two cysteine residues react with DTNB only after treatment with 8 m urea. The location of the various cysteine residues in the enzyme was established by using [¹⁴C]NEM followed by tryptic digestion and radiopeptide isolation. The reactive cysteine has been identified as Cys²⁹¹, and the thiol exposed with 3.5 m urea is Cys³⁴⁴. When Cys³⁴⁴ was replaced by either serine or alanine, the mutant enzymes were found to be as active as the wild-type enzyme. These findings point to the nonessential role of Cys³⁴⁴ in adenylosuccinate synthetase.