Arginine 132 (R132) mutations to histidine or cysteine frequently occur to cytosolic NADP+ -isocitrate dehydrogenase (IDH1) in secondary glioblastoma multiforme (GBM) patients, in which GBM develops from a lower grade astroctyoma. Mutant enzymes lose the normal IDH activity, but acquire a neomorphic ability of producing 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). In the present study, the analogous mutations, Arg to His or Cys, were employed to homologous Arg153 of the NADP+ -IDH from Escherichia coli (EcIDH), generating two mutants: EcIDH R153 H and EcIDH R153C. The mutations dramatically reduced the catalytic efficiencies (kcat /Km ) of EcIDH R153H and EcIDH R153C for isocitrate oxidation, which dropped to only 0.6 and 1.5% of the wild-type enzyme, respectively. Neoenzymatic activities of catalyzing α-KG to 2-HG by EcIDH R153H and EcIDH R153C were confirmed by GC/TOF-MS analysis. The Km values of EcIDH R153H and EcIDH R153C displayed for α-KG were 3.3 ± 0.12 and 2.2 ± 0.13 mM, respectively, and the catalytic efficiencies (kcat /Km ) of the two mutants for α-KG were 300 and 450 M-1 s-1 , respectively. As human IDH1 Arg132 mutation is cancer-associated, the present study provides new information for the in-depth investigation of the metabolic influence of EcIDH Arg mutation in vivo.
Keywords: Arginine mutation; Escherichia coli; GC; Isocitrate dehydrogenase; Neoenzymatic activity; TOF-MS.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.