Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase.
Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg(2+), Mn(2+ )or Fe(2+) ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.
Murakami
Keiko
K
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
Tsubouchi
Ryoko
R
Fukayama
Minoru
M
Ogawa
Tadashi
T
Yoshino
Masataka
M
D001665:Binding Sites / Q000235:genetics; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D005296:Ferrous Compounds / Q000494:pharmacology*; D005954:Glucosephosphate Dehydrogenase / Q000187:drug effects / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000494:pharmacology*
D003470:Culture Media; D029968:Escherichia coli Proteins; D005296:Ferrous Compounds; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase