Reversible modification of the sulfhydryl groups of Escherichia coli succinic thiokinase with methanethiolating reagents, 5,5′-dithio-bis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and ethylmercurithiosalicylate

Inhibition of Escherichia coli succinic thiokinase by either methyl methanethiolsulfonate (MeS-SO₂Me) or by methoxycarbonylmethyl disulfide was reversed completely by addition of tributylphosphine, but not by incubation with dithiothreitol. At a point where about 4 moles of MeS- were incorporated per mole of enzyme (M_r 140,000), no loss of antigenicity (as measured by microcomplement fixation) was observed, but at least 80% of thiokinase activity was lost. No significant conformational change was indicated for this methanethiolated enzyme in the ultracentrifuge. In contrast, losses of both thiokinase activity and antigenicity were brought about by incubation with 5,5′-dithiobis 2-nitrobenzoic acid) (Nbs₂), ethylmercurithiosalicylate, and p-hydroxymercuribenzoate. These losses were restored totally by incubation of modified enzyme with dithiothreitol but not with tributylphosphine. Preincubation of succinic thiokinases with MeSSO₂Me and with Nbs₂ protected the enzyme partially, and to the same degree, against the inhibiting effects of N-ethylmaleimide. These data suggest that, while both MeSSO₂Me and Nbs₂ may attack identical thiol groups on the enzyme, each may also react specifically with other thiol groups. Inhibition by MeS-SO₂Me and by Nbs₂ both appear not to be accompanied by dissociation into subunits and, in the later case, significant aggregation appears to be involved.