Immunochemical Studies of Escherichia coli Succinic Thiokinase

Abstract

Abstract Reaction of Escherichia coli succinic thiokinase [succinate:coenzyme A ligase (adenosine diphosphate) (EC 6.2.1.5)] with antiserum to the enzyme was monitored by microcomplement fixation analysis. Changes in the enzyme induced by heating, exposure to extremes of pH, treatment with urea, and incubation with p-mercuribenzoate (p-MB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and iodoacetamide were assayed by this technique. Horizontal shifts in the complement fixation curves obtained as a result of most of these treatments have been shown to be related quantitatively to the loss of enzymatic activity, except at low residual succinic thiokinase activities. Although enzymatic activity was almost completely lost in the case of the iodoacetamide-treated enzyme, the quaternary structure of the protein appeared to be maintained for a significant period of time. Vertical decreases in the complement fixation curves were interpreted to indicate dissociation into subunits. DTNB and p-MB titration of the enzyme have resulted in different vertical shifts of the complement fixation curves, and the plots of enzyme inactivation versus number of sulfhydryl groups titrated are different for these reagents. Titration of succinic thiokinase with DTNB in the presence of sodium dodecyl sulfate yielded 16 to 18 moles of sulfhydryl groups per 140,000 g of protein, in contradiction to previously published data.