THE REGULATION OF ESCHERICHIA-COLI GLUTAMINE-SYNTHETASE REVISITED - ROLE OF 2-KETOGLUTARATE IN THE REGULATION OF GLUTAMINE-SYNTHETASE ADENYLYLATION STATE

The regulation of Escherichia call glutamine synthetase (GS) by revers ible adenylylation has provided one of the classical paradigms for sig nal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation o f GS adenylylation state in a reconstituted system containing GS, aden ylyltransferase (ATase), the PII signal transduction protein that cont rols ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTas e/UR), which has a role in regulating PII. in this reconstituted bicyc lic cascade system, the adenylylation state of GS was regulated recipr ocally by the small molecule effecters 2-ketoglutarate and glutamine a t physiological effector concentrations. By examination of the individ ual regulatory monocycles and comparison to the bicyclic system and ex isting data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that th e main role of 2-ketoglutarate in bringing about the deadenylylation o f GS was to inhibit GS adenylylation, and this was due to the alloster ic regulation of PII activity. Glutamine acted as an allosteric regula tor of both ATase and UTase/UR. We also compared the regulation of GS adenylylation stale to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system con taining NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutar ate concentration, the regulation of GS adenylylation state by glutami ne was sharper and occurred at a higher concentration than did the reg ulation of NRI phosphorylation. The possible biological implications o f this regulatory arrangement are discussed.