Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase

Abstract

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.

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…In vitro protein oxidation systems have commonly used forcing circumstances to overcome these natural barriers. Efficient FeSOD inactivation, for example, requires supraphysiological levels of H 2 O 2 (100), and isocitrate dehydrogenase is damaged only when enough iron is added to out-compete manganese for the metal binding site (101). To date no metabolic pathway failures in moderately stressed cells have been linked to these types of metal-catalyzed oxidation reactions.…”

Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese

“…The metal coordinates substrate and stabilizes the intermediate oxyanion (25). (27), and the reduction of Fe by ascorbate regenerated Fe 2+ . At various time points the Fe and H 2 O 2 were removed by diethylenetriaminepentaacetic acid (DTPA) and catalase, and the amount of functional Rpe polypeptide was determined by the addition of Co 2+ before assay.…”

Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in Escherichia coli

“…The branch point at the TCA cycle and glyoxylate bypass is controlled by enzyme expression and depends on the growth conditions (16) and the NADP-isocitrate dehydrogenase activity (12), which is subject to inhibition by various metal ions (7,14,15) and phosphorylation/dephosphorylation of the protein (2,9,12). Here we report the inhibitory effects of PEP on the NADP-isocitrate dehydrogenase and isocitrate lyase purified from the archived clones of the icd-and aceAoverexpressing E. coli mutants (8), which were constructed with plasmid pCA24N from the E. coli strain K-12 W3110 (4).…”

“…The 1-ml reaction mixture contained 100 mM morpholinepropanesulfonic acid (MOPS) buffer (pH 7.1), 0.5 mM NADP, 0.5 mM MgCl 2 , and various concentrations of threo-DS-isocitrate in the absence and presence of phosphoenolpyruvate, and the activity was determined by monitoring the change in the absorbance at 340 nm (7,8). Plots of reaction velocity as a function of the substrate isocitrate concentration gave a hyperbolic curve, which became sigmoid in shape with the addition of PEP (Fig.…”