Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

• Published in: The Journal of biological chemistry Vol. 263; no. 5; pp. 2477 - 2482
• Main Authors: Robertson, E F, Hoyt, J C, Reeves, H C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.02.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Acid Phosphatase - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Diethyl Pyrocarbonate - pharmacology; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; histidine; Histidine - metabolism; isocitrate lyase; Isocitrate Lyase - metabolism; Isoelectric Point; Lyases; Oxo-Acid-Lyases - metabolism; Phosphorylation; Phosphorylation; Purification; Gel electrophoresis; Enzyme; Escherichia coli; Dephosphorylation; In vitro; Isocitrate lyase; Ion exchange chromatography; Autoradiography; Immunoprecipitation reaction; Chemical modification; Histidine; Bacteria; Escherichieae; ATP; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)69231-2

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.