title: Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability.
abstract: L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon. GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization. Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation. Purified GDH has a single tight site for NADPH binding. Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme. NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates. GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH. Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP. Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. mmaurizi@helix.nih.gov
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
authors.forename: Fatima
authors.initials: F
authors.lastname: Rasulova
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000494:Allosteric Regulation; D000641:Ammonia / Q000378:metabolism; D001222:Aspartate Kinase / Q000378:metabolism; D002264:Carboxylic Acids / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D003545:Cysteine; D049071:Endopeptidase Clp; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005947:Glucose / Q000172:deficiency; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D049070:Protease La; D011487:Protein Conformation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012697:Serine Endopeptidases / Q000378:metabolism; D051996:Transcription Factor RelA
chemical_list: D002264:Carboxylic Acids; C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D016328:NF-kappa B; D009711:Nucleotides; D051996:Transcription Factor RelA; D009249:NADP; D010664:Phenylmethylsulfonyl Fluoride; D002701:Chloramphenicol; D000641:Ammonia; D005969:Glutamate Dehydrogenase; D001222:Aspartate Kinase; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D005947:Glucose; D003545:Cysteine; D011190:Potassium Cyanide