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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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Related in: MedlinePlus

DON-treated CtpS is enzymatically inactive.Samples of CtpS were allowed to polymerize in activity buffer. Then 10 mM DON was added to stop enzymatic activity. (A and B) Two independent representative experiments are shown. Light scattering (polymerization) and transmittance (CTP production) are shown on the same axis in arbitrary units. (C) Polymerization and activity before and after DON addition are compared. Overall amplitude of light scattering and transmittance data is affected by absorption by DON at the wavelengths of light used for the assay. Therefore, the slopes of each condition are shown.DOI:http://dx.doi.org/10.7554/eLife.03638.015
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fig2s4: DON-treated CtpS is enzymatically inactive.Samples of CtpS were allowed to polymerize in activity buffer. Then 10 mM DON was added to stop enzymatic activity. (A and B) Two independent representative experiments are shown. Light scattering (polymerization) and transmittance (CTP production) are shown on the same axis in arbitrary units. (C) Polymerization and activity before and after DON addition are compared. Overall amplitude of light scattering and transmittance data is affected by absorption by DON at the wavelengths of light used for the assay. Therefore, the slopes of each condition are shown.DOI:http://dx.doi.org/10.7554/eLife.03638.015

Mentions: The CtpS crystal structure suggests that the enzyme's UTP and CTP binding sites partially overlap (Endrizzi et al., 2005), raising the question of whether CtpS assembly is controlled by the absolute level of CTP or the relative product/substrate levels. 6-Diazo-5-oxo-L-norleucine (DON) is a glutamine analog that covalently binds glutaminase active sites and irreversibly inactivates enzymatic activity (Chakraborty and Hurlbert, 1961). When added to activity buffer, DON abolishes both CTP production and CtpS polymerization (Figure 2—figure supplement 4). However, DON-treated CtpS can still polymerize when CTP is added to the solution (Figure 2E). Polymers formed in the presence of CTP and DON disassemble upon the addition of substrates but do not reform after substrate addition (Figure 2E), presumably because the DON-inhibited CtpS cannot produce additional CTP. DON treatment has no effect on CtpS polymerization when the enzyme is incubated with saturating CTP (Figure 2—figure supplements 1 and 5). These results suggest that competition between substrate (UTP) and product (CTP) binding controls the polymerization equilibrium of CtpS. The dependence of polymerization on CTP levels may explain why DON treatment abolishes in vivo CtpS assembly in some cellular contexts (Ingerson-Mahar et al., 2010) but not others (Chen et al., 2011).


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)

DON-treated CtpS is enzymatically inactive.Samples of CtpS were allowed to polymerize in activity buffer. Then 10 mM DON was added to stop enzymatic activity. (A and B) Two independent representative experiments are shown. Light scattering (polymerization) and transmittance (CTP production) are shown on the same axis in arbitrary units. (C) Polymerization and activity before and after DON addition are compared. Overall amplitude of light scattering and transmittance data is affected by absorption by DON at the wavelengths of light used for the assay. Therefore, the slopes of each condition are shown.DOI:http://dx.doi.org/10.7554/eLife.03638.015
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126345&req=5

fig2s4: DON-treated CtpS is enzymatically inactive.Samples of CtpS were allowed to polymerize in activity buffer. Then 10 mM DON was added to stop enzymatic activity. (A and B) Two independent representative experiments are shown. Light scattering (polymerization) and transmittance (CTP production) are shown on the same axis in arbitrary units. (C) Polymerization and activity before and after DON addition are compared. Overall amplitude of light scattering and transmittance data is affected by absorption by DON at the wavelengths of light used for the assay. Therefore, the slopes of each condition are shown.DOI:http://dx.doi.org/10.7554/eLife.03638.015
Mentions: The CtpS crystal structure suggests that the enzyme's UTP and CTP binding sites partially overlap (Endrizzi et al., 2005), raising the question of whether CtpS assembly is controlled by the absolute level of CTP or the relative product/substrate levels. 6-Diazo-5-oxo-L-norleucine (DON) is a glutamine analog that covalently binds glutaminase active sites and irreversibly inactivates enzymatic activity (Chakraborty and Hurlbert, 1961). When added to activity buffer, DON abolishes both CTP production and CtpS polymerization (Figure 2—figure supplement 4). However, DON-treated CtpS can still polymerize when CTP is added to the solution (Figure 2E). Polymers formed in the presence of CTP and DON disassemble upon the addition of substrates but do not reform after substrate addition (Figure 2E), presumably because the DON-inhibited CtpS cannot produce additional CTP. DON treatment has no effect on CtpS polymerization when the enzyme is incubated with saturating CTP (Figure 2—figure supplements 1 and 5). These results suggest that competition between substrate (UTP) and product (CTP) binding controls the polymerization equilibrium of CtpS. The dependence of polymerization on CTP levels may explain why DON treatment abolishes in vivo CtpS assembly in some cellular contexts (Ingerson-Mahar et al., 2010) but not others (Chen et al., 2011).

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
Related in: MedlinePlus