title: Iron-dependent oxidative inactivation with affinity cleavage of pyruvate kinase.
abstract: Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Murakami
authors.forename: Ryoko
authors.initials: R
authors.lastname: Tsubouchi
authors.forename: Minoru
authors.initials: M
authors.lastname: Fukayama
authors.forename: Shanlou
authors.initials: S
authors.lastname: Qiao
authors.forename: Masataka
authors.initials: M
authors.lastname: Yoshino
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007501:Iron / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007501:Iron; D011770:Pyruvate Kinase; D001205:Ascorbic Acid