Maurizi Michael R, Rasulova Fatima
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 2002 Jan 15;397(2):206-16. doi: 10.1006/abbi.2001.2703.
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.
L-谷氨酸脱氢酶（GDH）在指数生长的大肠杆菌细胞中是稳定的，但在缺乏氮或碳的饥饿细胞中以每小时20%-30%的速率降解。GDH的降解是能量依赖性的，ATP依赖性蛋白酶ClpAP或Lon中的突变导致部分稳定。降解受到氯霉素的抑制，并在relA突变细胞中完全被阻断，这表明核糖体介导信号传导可能促进GDH降解。纯化的GDH具有一个与NADPH结合的紧密位点。在没有其他配体的情况下，NADPH结合导致酶不稳定化作用增强。三羧酸及二羧酸或核苷二磷酸和三磷酸可逆转NADPH诱导的不稳定性和对蛋白水解的敏感性。GTP和ppGpp在变构位点与GDH结合，并逆转NADPH的不稳定化作用。天然GDH对几种纯化的ATP依赖性蛋白酶（ClpAP、ClpXP、Lon和ClpYQ）的降解具有抗性，但变性的GDH可被ClpAP降解。我们的结果表明，在体内，GDH因失去稳定配体或与饥饿细胞中积累的不稳定代谢物相互作用而对蛋白酶敏感，并且几种ATP依赖性蛋白酶中的任何一种都可降解这种敏感化蛋白质。
