title: Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes.
abstract: Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP(+) in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0-2.5mM, 2-3h, 37°C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500μM. Incubation with radiolabeled MGO (0-500µM, 0-3h, 37°C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP(+) binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.
authors.affiliation: Free Radical Group, The Heart Research Institute, Newtown, NSW 2042, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: morganp@hri.org.au.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Morgan
authors.affiliation: Free Radical Group, The Heart Research Institute, Newtown, NSW 2042, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Sheahan
authors.affiliation: Free Radical Group, The Heart Research Institute, Newtown, NSW 2042, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: David I
authors.initials: DI
authors.lastname: Pattison
authors.affiliation: Free Radical Group, The Heart Research Institute, Newtown, NSW 2042, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000096:biosynthesis*; D011765:Pyruvaldehyde / Q000494:pharmacology*
chemical_list: D017127:Glycation End Products, Advanced; D009249:NADP; D011765:Pyruvaldehyde; D001120:Arginine; D008291:Malate Dehydrogenase; C495394:malic enzyme 2; human; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase
keywords: Advanced glycation end-products; Arginine; Diabetes-associated atherosclerosis; Free radicals; Glycated proteins; Methylglyoxal; NADPH; Reactive aldehydes