Essential sulfhydryl group of malic enzyme from Escherichia coli.

The activity of malic enzyme from Escherichia coli was unaffected by the monovalent cations Na+ or Li+ at 10 mM. At 100 mM, Li+ or Na+ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40-80-fold with 10 mM K+, Rb+, Cs+, or NH4+. Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by dithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K+ stimulation, which suggested the essentiality of the sulfhydryl groups of the E. coli malic enzyme.