title: Mutations in human GLUD2 glutamate dehydrogenase affecting basal activity and regulation.
abstract: Glutamate dehydrogenase (GDH) in human exists in GLUD1 and GLUD2 gene-encoded isoforms (hGDH1 and hGDH2, respectively), differing in their regulation and tissue expression pattern. Whereas hGDH1 is subject to GTP control, hGDH2 uses for its regulation, a novel molecular mechanism not requiring GTP. This is based on the ability of hGDH2 to maintain a baseline activity of <10% of its capacity subject to full activation by rising ADP/L-leucine levels. Here we studied further the molecular mechanisms regulating hGDH2 function by creating and analyzing hGDH2 mutants harboring single amino acid substitutions in the regulatory domain (antenna, pivot helix) of the protein. Five hGDH2 mutants were obtained: two with an amino acid change (Gln441Arg, Ser445Leu) in the antenna, two (Lys450Glu, His454Tyr) in the pivot helix, and one (Ser448Pro) in the junction between the two structures. Functional analyses revealed that, while the antenna mutations increased basal enzyme activity without affecting its allosteric properties, the pivot helix mutations drastically reduced basal activity and impaired enzyme regulation. On the other hand, the Ser448Pro mutation reduced basal activity but did not alter allosteric regulation. Also, compared with wild-type hGDH2, the antenna mutants were relatively thermostable, whereas the pivot helix mutants were extremely heat labile. Hence, the present data further our understanding of the molecular mechanisms involved in the function and stability of hGDH2, an enzyme thought to be of importance for nerve tissue biology.
authors.affiliation: Department of Neurology, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Kanavouras
authors.forename: Nikolas
authors.initials: N
authors.lastname: Borompokas
authors.forename: Helen
authors.initials: H
authors.lastname: Latsoudis
authors.forename: Argiris
authors.initials: A
authors.lastname: Stagourakis
authors.forename: Ioannis
authors.initials: I
authors.lastname: Zaganas
authors.forename: Andreas
authors.initials: A
authors.lastname: Plaitakis
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D011487:Protein Conformation
chemical_list: D018076:DNA, Complementary; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase; C521120:GLUD2 protein, human