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Side‐chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation
Author(s) -
Mastorodemos Vasileios,
Kanavouras Konstantinos,
Sundaram Shobana,
Providaki Maria,
Petraki Zoe,
Kokkinidis Michael,
Zaganas Ioannis,
Logothetis Diomedes E.,
Plaitakis Andreas
Publication year - 2015
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13019
Subject(s) - glutamate receptor , biochemistry , biology , gtp' , mutation , leucine , mutant , microbiology and biotechnology , chemistry , amino acid , enzyme , receptor , gene
Glutamate Dehydrogenase ( GDH ) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system ( CNS ). hGDH 1 is activated by ADP and L‐leucine and powerfully inhibited by GTP . Besides this housekeeping hGDH 1, duplication led to an hGDH 2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4–6% of capacity) while remaining remarkably responsive to ADP /L‐leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH 1 is shown to diminish basal activity (< 2% of capacity) and abrogate L‐leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain (‘antenna’), we replaced Ser409 by Arg or Asp in hGDH 1. The Ser409Arg‐1 change essentially replicated the Arg443Ser‐1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443‐1 or Arg409‐1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser‐1 mutant protein, obtained in recombinant form, regained most of the wild‐type hGDH 1 properties. Also, when Ser443 was replaced by Arg443 in hGDH 2 (as occurs in hGDH 1), the Ser443Arg‐2 mutant acquired most of the hGDH 1 properties. Hence, side‐chain interactions between 409 and 443 positions in the ‘antenna’ region of hGDH s are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.Glutamate dehydrogenase (GDH) is central to the metabolism of the excitatory transmitter glutamate, and links it with carbohydrate metabolism in energy homeostasis and cell signaling. The isoform hGDH2, in contrast to hGDH1, is dissociated from GTP (guanosine triphosphate) control, has a reduced basal activity, but remains highly responsive to ADP/L‐leucine activation. Substitution of Serine (Ser) for arginine (Arg443) in hGDH1 diminishes basal activity (< 2% of capacity) and abrogates L‐leucine activation. We provide evidence that side‐chain interactions between 409 and 443 positions in the regulatory domain of GDH are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.