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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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Polymerization enhances the inhibition of CtpS activity by CTP.At a CtpS concentration below the threshold concentration, (200 nM, red circles), the CTP IC50 value is 330 μM. At concentrations that favor polymerization (4 μM CtpS, green squares), CTP binds with higher apparent affinity with an IC50 of 170 μM. Abolishing polymerization with E277R mutation reduced apparent CTP activity inhibtion (IC50 = 833 μM at 200 nM CtpSE277R) (purple open diamonds). The CTP synthesis vo values before normalization were 1.24, 18.5, and 0.82 μM/s for 200 nM CtpS, 4 μM CtpS, and 200 nM CtpSE277R, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.026
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fig7s2: Polymerization enhances the inhibition of CtpS activity by CTP.At a CtpS concentration below the threshold concentration, (200 nM, red circles), the CTP IC50 value is 330 μM. At concentrations that favor polymerization (4 μM CtpS, green squares), CTP binds with higher apparent affinity with an IC50 of 170 μM. Abolishing polymerization with E277R mutation reduced apparent CTP activity inhibtion (IC50 = 833 μM at 200 nM CtpSE277R) (purple open diamonds). The CTP synthesis vo values before normalization were 1.24, 18.5, and 0.82 μM/s for 200 nM CtpS, 4 μM CtpS, and 200 nM CtpSE277R, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.026

Mentions: Whereas CtpSE277R was slightly impaired in its activity at low enzyme concentrations, CtpSE277R exhibited a much higher concentration at which kcat is one half of its maximum due to polymerization (the [CtpS]0.5 value) compared to wild-type CtpS ([CtpSE277R]0.5 = 7.1 μM vs [CtpS]0.5 = 3.3 μM). Furthermore, the concentration dependence of CtpSE277Rkcat was less steep than wild type, with CtpSE277R retaining 48% of its maximal activity at the highest enzyme concentration tested (8 μM) (Figure 7A). This behavior was in stark contrast to wild-type CtpS, whose activity plummeted to 4% of its maximum. Thus, at low enzyme concentrations, CtpSE277R exhibited slightly lower activity than wild type while at high enzyme concentrations CtpSE277R activity was significantly greater than that of wild type. One explanation for the comparatively modest decrease in CtpSE277R activity as a function of enzyme concentration is that CtpSE277R produces CTP, which at high CtpS concentrations can accumulate and competitively inhibit CtpS activity, resulting in a slight activity decrease. However, this mutant lacks the dramatic reduction in CtpS activity mediated by large-scale assembly into filaments. As predicted from thermodynamic linkage, the inability to polymerize also leads CtpSE77R to bind CTP less tightly, with a higher IC50 value than the wild-type enzyme (830 μM vs 360 μM at 200 nM enzyme, Figure 7—figure supplement 2). These data are thus consistent with the model that CtpS is negatively regulated in two ways: CTP competitively inhibits UTP binding, and large-scale assembly sterically hinders a conformational change required for CtpS activity. The quantitative differences between wild type and CtpSE277R activity suggest that large-scale assembly mediates rapid and efficient inhibition of enzymatic activity.


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)

Polymerization enhances the inhibition of CtpS activity by CTP.At a CtpS concentration below the threshold concentration, (200 nM, red circles), the CTP IC50 value is 330 μM. At concentrations that favor polymerization (4 μM CtpS, green squares), CTP binds with higher apparent affinity with an IC50 of 170 μM. Abolishing polymerization with E277R mutation reduced apparent CTP activity inhibtion (IC50 = 833 μM at 200 nM CtpSE277R) (purple open diamonds). The CTP synthesis vo values before normalization were 1.24, 18.5, and 0.82 μM/s for 200 nM CtpS, 4 μM CtpS, and 200 nM CtpSE277R, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.026
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126345&req=5

fig7s2: Polymerization enhances the inhibition of CtpS activity by CTP.At a CtpS concentration below the threshold concentration, (200 nM, red circles), the CTP IC50 value is 330 μM. At concentrations that favor polymerization (4 μM CtpS, green squares), CTP binds with higher apparent affinity with an IC50 of 170 μM. Abolishing polymerization with E277R mutation reduced apparent CTP activity inhibtion (IC50 = 833 μM at 200 nM CtpSE277R) (purple open diamonds). The CTP synthesis vo values before normalization were 1.24, 18.5, and 0.82 μM/s for 200 nM CtpS, 4 μM CtpS, and 200 nM CtpSE277R, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.026
Mentions: Whereas CtpSE277R was slightly impaired in its activity at low enzyme concentrations, CtpSE277R exhibited a much higher concentration at which kcat is one half of its maximum due to polymerization (the [CtpS]0.5 value) compared to wild-type CtpS ([CtpSE277R]0.5 = 7.1 μM vs [CtpS]0.5 = 3.3 μM). Furthermore, the concentration dependence of CtpSE277Rkcat was less steep than wild type, with CtpSE277R retaining 48% of its maximal activity at the highest enzyme concentration tested (8 μM) (Figure 7A). This behavior was in stark contrast to wild-type CtpS, whose activity plummeted to 4% of its maximum. Thus, at low enzyme concentrations, CtpSE277R exhibited slightly lower activity than wild type while at high enzyme concentrations CtpSE277R activity was significantly greater than that of wild type. One explanation for the comparatively modest decrease in CtpSE277R activity as a function of enzyme concentration is that CtpSE277R produces CTP, which at high CtpS concentrations can accumulate and competitively inhibit CtpS activity, resulting in a slight activity decrease. However, this mutant lacks the dramatic reduction in CtpS activity mediated by large-scale assembly into filaments. As predicted from thermodynamic linkage, the inability to polymerize also leads CtpSE77R to bind CTP less tightly, with a higher IC50 value than the wild-type enzyme (830 μM vs 360 μM at 200 nM enzyme, Figure 7—figure supplement 2). These data are thus consistent with the model that CtpS is negatively regulated in two ways: CTP competitively inhibits UTP binding, and large-scale assembly sterically hinders a conformational change required for CtpS activity. The quantitative differences between wild type and CtpSE277R activity suggest that large-scale assembly mediates rapid and efficient inhibition of enzymatic activity.

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