title: Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate.
abstract: Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the "ThDP-enamine"/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an "off-pathway" side reaction comprising less than 1% of the "on-pathway" reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Nemeria
authors.affiliation: the Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest 1094, Hungary, and.
authors.forename: Attila
authors.initials: A
authors.lastname: Ambrus
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Hetalben
authors.initials: H
authors.lastname: Patel
authors.affiliation: the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Gary
authors.initials: G
authors.lastname: Gerfen
authors.affiliation: the Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest 1094, Hungary, and.
authors.forename: Vera
authors.initials: V
authors.lastname: Adam-Vizi
authors.affiliation: the Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest 1094, Hungary, and.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Tretter
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Jieyu
authors.initials: J
authors.lastname: Zhou
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Junjie
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102, frjordan@rutgers.edu.
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D000588:Amines / Q000378:metabolism*; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007656:Ketoglutaric Acids; D007700:Kinetics; D063065:Organophosphonates / Q000378:metabolism; D010084:Oxidation-Reduction; D010721:Phosphinic Acids / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013057:Spectrum Analysis; D013831:Thiamine; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D000588:Amines; D005609:Free Radicals; D007656:Ketoglutaric Acids; D063065:Organophosphonates; D010721:Phosphinic Acids; D017382:Reactive Oxygen Species; D007655:Ketoglutarate Dehydrogenase Complex; D013835:Thiamine Pyrophosphate; D013831:Thiamine
keywords: Dehydrogenase; Electron Paramagnetic Resonance (EPR); Mass Spectrometry (MS); Reactive Oxygen Species (ROS); Thiamin