Reconstitution of Escherichia coli glutamine synthetase adenylyltransferase from N-terminal and C-terminal fragments of the enzyme.
ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adenylylation and deadenylylation of GS in response to signals of cellular nitrogen status and energy. The adenylyltransferase (AT) activity of ATase is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by PII-UMP. Conversely, the adenylyl-removing (AR) activity of ATase is activated by PII-UMP and inhibited by unmodified PII and by glutamine. Here, we show that the enzyme can be reconstituted from two purified polypeptides that comprise the N-terminal two-thirds of the protein and the C-terminal one-third of the protein. Properties of the reconstituted enzyme support recent hypotheses for the sites of regulatory interactions and mechanisms for intramolecular signal transduction. Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain. Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains. Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.
Jiang
Peng
P
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu
Ninfa
Alexander J
AJ
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments; D005973:Glutamine; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase