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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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PII activation of the reconstituted enzyme displays synergy with glutamine and biphasic sensitivity to α-ketoglutarate, as observed with the wild-type enzyme. (A) Activation of the AT activity of the reconstituted enzyme by glutamine. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 0.05 μM, ATC3 at 0.25 μM, ATN6 at 10 μM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. As shown, the apparent Kact for glutamine was ∼7 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (B) Activation of the AT activity of the reconstituted enzyme by glutamine in the presence of a saturating concentration of PII. Conditions were as described for panel C, except that PII was present at 15 μM and ATC3 was present at 0.05 μM. As shown, the apparent Kact for glutamine was ∼0.55 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (C) Biphasic effects of α-ketoglutarate on the activation of the AT activity of the reconstituted enzyme. Conditions were as described for panel A, with GS at 2.5 μM, ATP at 0.5 mM, ATC3 at 1.6 μM, ATN6 at 5 μM, PII at 5 μM, PK at 0.022 unit/μL, and PEP at 5 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%.
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fig4: PII activation of the reconstituted enzyme displays synergy with glutamine and biphasic sensitivity to α-ketoglutarate, as observed with the wild-type enzyme. (A) Activation of the AT activity of the reconstituted enzyme by glutamine. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 0.05 μM, ATC3 at 0.25 μM, ATN6 at 10 μM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. As shown, the apparent Kact for glutamine was ∼7 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (B) Activation of the AT activity of the reconstituted enzyme by glutamine in the presence of a saturating concentration of PII. Conditions were as described for panel C, except that PII was present at 15 μM and ATC3 was present at 0.05 μM. As shown, the apparent Kact for glutamine was ∼0.55 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (C) Biphasic effects of α-ketoglutarate on the activation of the AT activity of the reconstituted enzyme. Conditions were as described for panel A, with GS at 2.5 μM, ATP at 0.5 mM, ATC3 at 1.6 μM, ATN6 at 5 μM, PII at 5 μM, PK at 0.022 unit/μL, and PEP at 5 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%.

Mentions: In Figure 3, it was observed that the ATN6 polypeptide mediated the activation of the AT activity by PII and that this activation was synergistic with activation by glutamine. To examine this synergy in more detail, we measured the glutamine Kact for the reconstituted enzyme in the presence and absence of PII. The wild-type enzyme displays strong synergy in activation by PII and glutamine; the presence of either of these activators dramatically reduced the Kact for activation by the other activator (9). To ensure that the ATC3 polypeptide was mostly contained within the reconstituted enzyme, these experiments were conducted under conditions with a large excess of the ATN6 polypeptide (Figure 4). The reconstitited enzyme exhibited a glutamine Kact of ∼ 7 mM (Figure 4A), similar to the that of intact enzyme (9) and higher than the 1.5 mM glutamine Kact of the ATC3 polypeptide (10). When PII was present at a saturating concentration (15 μM), the glutamine Kact of the reconstituted enzyme was ∼0.55 mM (Figure 4B), remarkably reminiscent of values obtained for the wild-type enzyme of 0.45−0.5 mM (9,10). Thus, it appears that the synergy between glutamine and PII exhibited by the intact enzyme was also displayed, essentially in full measure, by the reconstituted enzyme.


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

Jiang P, Ninfa AJ - Biochemistry (2009)

PII activation of the reconstituted enzyme displays synergy with glutamine and biphasic sensitivity to α-ketoglutarate, as observed with the wild-type enzyme. (A) Activation of the AT activity of the reconstituted enzyme by glutamine. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 0.05 μM, ATC3 at 0.25 μM, ATN6 at 10 μM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. As shown, the apparent Kact for glutamine was ∼7 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (B) Activation of the AT activity of the reconstituted enzyme by glutamine in the presence of a saturating concentration of PII. Conditions were as described for panel C, except that PII was present at 15 μM and ATC3 was present at 0.05 μM. As shown, the apparent Kact for glutamine was ∼0.55 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (C) Biphasic effects of α-ketoglutarate on the activation of the AT activity of the reconstituted enzyme. Conditions were as described for panel A, with GS at 2.5 μM, ATP at 0.5 mM, ATC3 at 1.6 μM, ATN6 at 5 μM, PII at 5 μM, PK at 0.022 unit/μL, and PEP at 5 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651632&req=5

fig4: PII activation of the reconstituted enzyme displays synergy with glutamine and biphasic sensitivity to α-ketoglutarate, as observed with the wild-type enzyme. (A) Activation of the AT activity of the reconstituted enzyme by glutamine. Conditions were as described in , with GS at 2.5 μM, ATP at 0.5 mM, α-ketoglutarate at 0.05 μM, ATC3 at 0.25 μM, ATN6 at 10 μM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. As shown, the apparent Kact for glutamine was ∼7 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (B) Activation of the AT activity of the reconstituted enzyme by glutamine in the presence of a saturating concentration of PII. Conditions were as described for panel C, except that PII was present at 15 μM and ATC3 was present at 0.05 μM. As shown, the apparent Kact for glutamine was ∼0.55 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%. (C) Biphasic effects of α-ketoglutarate on the activation of the AT activity of the reconstituted enzyme. Conditions were as described for panel A, with GS at 2.5 μM, ATP at 0.5 mM, ATC3 at 1.6 μM, ATN6 at 5 μM, PII at 5 μM, PK at 0.022 unit/μL, and PEP at 5 mM. In general, errors for these types of experiments in our laboratory are typically on the order of 10−15%.
Mentions: In Figure 3, it was observed that the ATN6 polypeptide mediated the activation of the AT activity by PII and that this activation was synergistic with activation by glutamine. To examine this synergy in more detail, we measured the glutamine Kact for the reconstituted enzyme in the presence and absence of PII. The wild-type enzyme displays strong synergy in activation by PII and glutamine; the presence of either of these activators dramatically reduced the Kact for activation by the other activator (9). To ensure that the ATC3 polypeptide was mostly contained within the reconstituted enzyme, these experiments were conducted under conditions with a large excess of the ATN6 polypeptide (Figure 4). The reconstitited enzyme exhibited a glutamine Kact of ∼ 7 mM (Figure 4A), similar to the that of intact enzyme (9) and higher than the 1.5 mM glutamine Kact of the ATC3 polypeptide (10). When PII was present at a saturating concentration (15 μM), the glutamine Kact of the reconstituted enzyme was ∼0.55 mM (Figure 4B), remarkably reminiscent of values obtained for the wild-type enzyme of 0.45−0.5 mM (9,10). Thus, it appears that the synergy between glutamine and PII exhibited by the intact enzyme was also displayed, essentially in full measure, by the reconstituted enzyme.

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