title: The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases.
abstract: Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.
authors: Neurology Laboratory, Medical School, University of Crete, Heraklion, Crete, Greece. johnzag@yahoo.com
authors: Ioannis
authors: I
authors: Zaganas
authors: Kamilla
authors: K
authors: Pajęcka
authors: Camilla
authors: C
authors: Wendel Nielsen
authors: Arne
authors: A
authors: Schousboe
authors: Helle S
authors: HS
authors: Waagepetersen
authors: Andreas
authors: A
authors: Plaitakis
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism; D009249:NADP / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D007656:Ketoglutaric Acids; D009249:NADP; D000244:Adenosine Diphosphate; D000641:Ammonia; D005969:Glutamate Dehydrogenase