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The Effects of Nucleotides on the Conformational Flexibility and Stability of Glutamate Dehydrogenase
Author(s) -
Tran Sirena,
Bell Jessica K.,
Bell Ellis
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.762.8
Subject(s) - gtp' , allosteric regulation , chemistry , glutamate dehydrogenase , cooperativity , nad+ kinase , active site , nucleotide , biochemistry , binding site , oxidative deamination , conformational change , cooperative binding , cofactor , biophysics , enzyme , glutamate receptor , biology , receptor , gene
Glutamate Dehydrogenase (GDH) catalyzes the reversible oxidative deamination of glutamate using NAD(P) + (H), and is involved in various metabolic pathways and energy cycles. GDH can be allosterically regulated by various nucleotides (ADP, GTP) as well as showing cofactor cooperativity. Previous work shows NADH binds to both the active site and a second regulatory (inhibitory) site while NADPH binds only to the active site. We hypothesize that changes in stability/flexibility correlate with inhibition and activation. To explore the allosteric mode for GDH, we have used Fluorescence‐based Thermal Shift Assays (FTSA), Circular Dichroism (CD), Thermal melts (Tm) and Limited Proteolysis using immobilized trypsin and tandem mass spectrometry (LP) to explore the stability (global and localized) of the protein in the presence or absence of various combinations of nucleotides and substrates (glutamate or 2‐oxo‐glutarate). CD and Tm studies indicate that while the overall structure of the protein is not changed by pH, the stability changes dramatically over the pH range 6–8.5 with a pKa of 7.4. ADP stabilizes the protein while GTP destabilizes the protein, with coligands having little effect. NADPH shows a small destabilizing effect, while NADH effects are complex depending upon concentration. Below the pKa, FbTSA experiments show that ADP and NADH binding to its regulatory site stabilize the protein significantly, while NAD(P)H binding to the active site, or GTP binding destabilize the protein, consistent with the conclusions reached from the CD thermal melt studies. With ADP, binding curves established by FTSA indicate biphasic binding in the presence or absence of cofactor bound at the active site suggesting negative cooperativity in ADP binding. The presence of glutamate has little effect. At pH 8, ADP and GTP have similar effects, while with NADH and NADPH destabilizes the protein. To determine localized regions of the protein responsible for these effects, we are using LP experiments and mapping regions of flexibility changes on the overall 3 dimensional structure of the protein. Support or Funding Information Work was funded by the USD Knapp Chair and the USD McNair program.