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Iron-Dependent Oxidative Inactivation with Affinity Cleavage of Pyruvate Kinase

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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.

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Author notes

  1. Masataka Yoshino

    Present address: Department of Food and Nutritional Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya, 463-8521, Japan

Authors and Affiliations

  1. Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, 480-1195, Japan

    Keiko Murakami, Ryoko Tsubouchi & Masataka Yoshino

  2. Central Research Laboratory, Aichi Medical University School of Medicine, Nagkaute, Aichi, 480-1195, Japan

    Minoru Fukayama

  3. Division of Pathology and Histology, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, 1200 Matsumoto Cho, Kasugai, Aichi, 487-8501, Japan

    Shanlou Qiao

Authors
  1. Keiko Murakami
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  2. Ryoko Tsubouchi
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  3. Minoru Fukayama
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  4. Shanlou Qiao
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  5. Masataka Yoshino
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Correspondence to Masataka Yoshino.

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Murakami, K., Tsubouchi, R., Fukayama, M. et al. Iron-Dependent Oxidative Inactivation with Affinity Cleavage of Pyruvate Kinase. Biol Trace Elem Res 130, 31–38 (2009). https://doi.org/10.1007/s12011-009-8317-x

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  • DOI: https://doi.org/10.1007/s12011-009-8317-x

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