title: Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate.
abstract: The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP. In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used. The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate. Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated. In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP. The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed. The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate.
authors.affiliation: Dipartimento di Biochimica, Università di Pavia.
authors.forename: G
authors.initials: G
authors.lastname: Valentini
authors.forename: M L
authors.initials: ML
authors.lastname: Speranza
authors.forename: P
authors.initials: P
authors.lastname: Iadarola
authors.forename: G
authors.initials: G
authors.lastname: Ferri
authors.forename: M
authors.initials: M
authors.lastname: Malcovati
mesh_terms: D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D014357:Trypsin
chemical_list: D005635:Fructosediphosphates; D006598:Hexosediphosphates; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D011770:Pyruvate Kinase; D014357:Trypsin; C029063:fructose-1,6-diphosphate