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Reconstitution of Escherichia coli glutamine synthetase adenylyltransferase from N-terminal and C-terminal fragments of the enzyme.

Jiang P, Ninfa AJ - Biochemistry (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu

ABSTRACT
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.

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ATN6 mediated the regulation of ATC3 by PII, regulated the basal AT activity of ATC3, and mediated inhibition of the ATC3 AT activity by PII-UMP. (A) ATN6 mediates PII regulation of ATC3 AT activity. The initial rate of GS adenylylation was measured as described in , with GS at 2.5 μM, α-ketoglutarate at 0.05 mM, [α-32P]ATP at 0.5 mM, pyruvate kinase at 0.022 unit/μL, PEP at 3 mM, bovine serum albumin at 0.3 mg/mL, and ATC3 at 0.25 μM. (B) ATN6 inhibits the basal AT activity of ATC3 and mediates PII activation of the ATC3 AT activity. Conditions were as described in  and for panel A, except that the concentration of the enzyme ATC3 was increased to 0.8 μM to facilitate examination of the basal AT activity. (C) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at the Kact. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 1 mM, ATC3 at 0.025 μM, glutamine at 1.5 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at a saturating level. Conditions were as described in  and for panel C, except that glutamine was present at a concentration of 20 mM and ATC3 was present at a concentration of 0.125 μM.
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fig3: ATN6 mediated the regulation of ATC3 by PII, regulated the basal AT activity of ATC3, and mediated inhibition of the ATC3 AT activity by PII-UMP. (A) ATN6 mediates PII regulation of ATC3 AT activity. The initial rate of GS adenylylation was measured as described in , with GS at 2.5 μM, α-ketoglutarate at 0.05 mM, [α-32P]ATP at 0.5 mM, pyruvate kinase at 0.022 unit/μL, PEP at 3 mM, bovine serum albumin at 0.3 mg/mL, and ATC3 at 0.25 μM. (B) ATN6 inhibits the basal AT activity of ATC3 and mediates PII activation of the ATC3 AT activity. Conditions were as described in and for panel A, except that the concentration of the enzyme ATC3 was increased to 0.8 μM to facilitate examination of the basal AT activity. (C) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at the Kact. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 1 mM, ATC3 at 0.025 μM, glutamine at 1.5 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at a saturating level. Conditions were as described in and for panel C, except that glutamine was present at a concentration of 20 mM and ATC3 was present at a concentration of 0.125 μM.

Mentions: Unlike wild-type ATase, the ATC3 polypeptide had basal AT activity in the absence of any activators (10) (Figure 3). This activity was strongly inhibited by the ATN6 polypeptide, which when present at a saturating concentration almost completely inhibited the basal AT activity (Figure 3). As before, the ATC3 polypeptide was activated by glutamine (10) (Figure 3), and we observed that AT activity in the presence of both glutamine and ATN6 was ∼5-fold higher than the strongly inhibited rate obtained when ATN6 was present in the absence of glutamine (Figure 3A).


Reconstitution of Escherichia coli glutamine synthetase adenylyltransferase from N-terminal and C-terminal fragments of the enzyme.

Jiang P, Ninfa AJ - Biochemistry (2009)

ATN6 mediated the regulation of ATC3 by PII, regulated the basal AT activity of ATC3, and mediated inhibition of the ATC3 AT activity by PII-UMP. (A) ATN6 mediates PII regulation of ATC3 AT activity. The initial rate of GS adenylylation was measured as described in , with GS at 2.5 μM, α-ketoglutarate at 0.05 mM, [α-32P]ATP at 0.5 mM, pyruvate kinase at 0.022 unit/μL, PEP at 3 mM, bovine serum albumin at 0.3 mg/mL, and ATC3 at 0.25 μM. (B) ATN6 inhibits the basal AT activity of ATC3 and mediates PII activation of the ATC3 AT activity. Conditions were as described in  and for panel A, except that the concentration of the enzyme ATC3 was increased to 0.8 μM to facilitate examination of the basal AT activity. (C) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at the Kact. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 1 mM, ATC3 at 0.025 μM, glutamine at 1.5 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at a saturating level. Conditions were as described in  and for panel C, except that glutamine was present at a concentration of 20 mM and ATC3 was present at a concentration of 0.125 μM.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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fig3: ATN6 mediated the regulation of ATC3 by PII, regulated the basal AT activity of ATC3, and mediated inhibition of the ATC3 AT activity by PII-UMP. (A) ATN6 mediates PII regulation of ATC3 AT activity. The initial rate of GS adenylylation was measured as described in , with GS at 2.5 μM, α-ketoglutarate at 0.05 mM, [α-32P]ATP at 0.5 mM, pyruvate kinase at 0.022 unit/μL, PEP at 3 mM, bovine serum albumin at 0.3 mg/mL, and ATC3 at 0.25 μM. (B) ATN6 inhibits the basal AT activity of ATC3 and mediates PII activation of the ATC3 AT activity. Conditions were as described in and for panel A, except that the concentration of the enzyme ATC3 was increased to 0.8 μM to facilitate examination of the basal AT activity. (C) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at the Kact. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 1 mM, ATC3 at 0.025 μM, glutamine at 1.5 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) ATN6 mediates PII-UMP inhibition of the AT activity of ATC3 when glutamine was at a saturating level. Conditions were as described in and for panel C, except that glutamine was present at a concentration of 20 mM and ATC3 was present at a concentration of 0.125 μM.
Mentions: Unlike wild-type ATase, the ATC3 polypeptide had basal AT activity in the absence of any activators (10) (Figure 3). This activity was strongly inhibited by the ATN6 polypeptide, which when present at a saturating concentration almost completely inhibited the basal AT activity (Figure 3). As before, the ATC3 polypeptide was activated by glutamine (10) (Figure 3), and we observed that AT activity in the presence of both glutamine and ATN6 was ∼5-fold higher than the strongly inhibited rate obtained when ATN6 was present in the absence of glutamine (Figure 3A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus