title: Isolation of pyrophosphohistidine from pyrophosphorylated pyruvate, phosphate dikinase.
abstract: The pyrophosphoryl form of pyruvate, phosphate dikinase was prepared by incubation with adenosine 5'-[gamma-32P]triphosphate and isolated by gel chromatography. Previously a phosphorylated moiety had been isolated from the enzyme and was shown to be bound through a phosphoramidate linkage to the 3' nitrogen of a histidine residue [Spronk, A. M., Yoshida, H., & Wood, H. G. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4415]. This histidine residue has been considered to be the pyrophosphoryl and phosphoryl carrier between the three subsites of this enzyme. Previous attempts to isolate the putative [32P]pyrophosphohistidine have been unsuccessful due to the lability of the [32P]pyrophosphoryl-enzyme. By stabilization of the [32P]pyrophosphoryl-enzyme with diazomethane, it has been possible to isolate a [32P]-pyrophosphohistidine from the hydrolysates. To our knowledge this work constitutes the first direct demonstration of a pyrophosphorylated histidyl residue in an enzyme.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D006639:Histidine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology; D011485:Protein Binding; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; C049223:pyrophosphohistidine; D006639:Histidine; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011772:Pyruvate, Orthophosphate Dikinase