Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

NADP+-specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with (32P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32P-labeled IDH was isolated from sonicated extracts of the cells. The 32P-enzyme was carboxylmethylated and digested with trypsin. A single 32P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue