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Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

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CtpS enzymatic activity or CTP addition is required for CtpS polymerization.Addition of various factors (shown in table) to 10 µM CtpS indicates that glutamine binding is dispensable for CtpS polymerization. Full activity buffer (containing 1 mM ATP, 1 mM UTP, and 10 mM glutamine) or CTP are required for polymerization. Omission of a substrate from activity buffer or addition of the inhibitor 1 mM DON inhibits polymerization. 1 mM DON has no effect on CTP-induced polymerization. Error bars = SE (n = 2).DOI:http://dx.doi.org/10.7554/eLife.03638.012
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fig2s1: CtpS enzymatic activity or CTP addition is required for CtpS polymerization.Addition of various factors (shown in table) to 10 µM CtpS indicates that glutamine binding is dispensable for CtpS polymerization. Full activity buffer (containing 1 mM ATP, 1 mM UTP, and 10 mM glutamine) or CTP are required for polymerization. Omission of a substrate from activity buffer or addition of the inhibitor 1 mM DON inhibits polymerization. 1 mM DON has no effect on CTP-induced polymerization. Error bars = SE (n = 2).DOI:http://dx.doi.org/10.7554/eLife.03638.012

Mentions: In order to identify the factors that control CtpS inhibition by assembly, we first confirmed that none of the substrates alone induced polymerization (Figure 2—figure supplement 1). We then directly tested our hypothesis that CtpS's product, CTP, a known inhibitor of CtpS activity, stimulates CtpS polymerization. In the absence of substrates (UTP, ATP, and glutamine), incubation with CTP caused CtpS to polymerize (Figure 2A). The threshold concentration for robust changes in light scattering by CtpS with saturating CTP (1–2 μM CtpS; Figure 2—figure supplement 2) agrees with the threshold concentration in the presence of substrates (1–2 μM CtpS; Figure 1—figure supplement 1). This result suggests that CTP alone is sufficient to influence polymerization and that the substrates and any other products of the enzymatic reaction are not necessary. To confirm that CTP stimulates CtpS assembly, we used ultracentrifugation as an independent assembly assay. Titrating with increasing amounts of CTP caused an increase in the amount of CtpS found in the pellet with respect to the 0 mM CTP condition (Figure 2B, Figure 2—figure supplement 3).10.7554/eLife.03638.011Figure 2.CTP is sufficient and necessary to stimulate CtpS polymerization.


Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

CtpS enzymatic activity or CTP addition is required for CtpS polymerization.Addition of various factors (shown in table) to 10 µM CtpS indicates that glutamine binding is dispensable for CtpS polymerization. Full activity buffer (containing 1 mM ATP, 1 mM UTP, and 10 mM glutamine) or CTP are required for polymerization. Omission of a substrate from activity buffer or addition of the inhibitor 1 mM DON inhibits polymerization. 1 mM DON has no effect on CTP-induced polymerization. Error bars = SE (n = 2).DOI:http://dx.doi.org/10.7554/eLife.03638.012
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126345&req=5

fig2s1: CtpS enzymatic activity or CTP addition is required for CtpS polymerization.Addition of various factors (shown in table) to 10 µM CtpS indicates that glutamine binding is dispensable for CtpS polymerization. Full activity buffer (containing 1 mM ATP, 1 mM UTP, and 10 mM glutamine) or CTP are required for polymerization. Omission of a substrate from activity buffer or addition of the inhibitor 1 mM DON inhibits polymerization. 1 mM DON has no effect on CTP-induced polymerization. Error bars = SE (n = 2).DOI:http://dx.doi.org/10.7554/eLife.03638.012
Mentions: In order to identify the factors that control CtpS inhibition by assembly, we first confirmed that none of the substrates alone induced polymerization (Figure 2—figure supplement 1). We then directly tested our hypothesis that CtpS's product, CTP, a known inhibitor of CtpS activity, stimulates CtpS polymerization. In the absence of substrates (UTP, ATP, and glutamine), incubation with CTP caused CtpS to polymerize (Figure 2A). The threshold concentration for robust changes in light scattering by CtpS with saturating CTP (1–2 μM CtpS; Figure 2—figure supplement 2) agrees with the threshold concentration in the presence of substrates (1–2 μM CtpS; Figure 1—figure supplement 1). This result suggests that CTP alone is sufficient to influence polymerization and that the substrates and any other products of the enzymatic reaction are not necessary. To confirm that CTP stimulates CtpS assembly, we used ultracentrifugation as an independent assembly assay. Titrating with increasing amounts of CTP caused an increase in the amount of CtpS found in the pellet with respect to the 0 mM CTP condition (Figure 2B, Figure 2—figure supplement 3).10.7554/eLife.03638.011Figure 2.CTP is sufficient and necessary to stimulate CtpS polymerization.

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

Show MeSH