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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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CTP binding enhances polymerization with a sharp response.The concentration required to reduce CtpS-specific activity (kcat) to 50% of its maximum value, [CtpS]0.5, is inversely related to the affinity of the polymer for Ctps tetramers. In the absence of CTP, the [CtpS]0.5 value is 3.3 μM (red circles). At a CTP concentration near the IC50 value (400 μM), the [CtpS]0.5 value is slightly reduced (2.8 μM, green squares), while at 800 μM, the [CtpS]0.5 value significantly shifted towards polymerization ([CtpS]0.5 = 1.4 μM, blue open diamonds). The maximum kcat values before normalization were 6.7, 3.5, and 1.04 s−1 for experiments using 0, 400, and 800 μM CTP, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.032
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fig8s3: CTP binding enhances polymerization with a sharp response.The concentration required to reduce CtpS-specific activity (kcat) to 50% of its maximum value, [CtpS]0.5, is inversely related to the affinity of the polymer for Ctps tetramers. In the absence of CTP, the [CtpS]0.5 value is 3.3 μM (red circles). At a CTP concentration near the IC50 value (400 μM), the [CtpS]0.5 value is slightly reduced (2.8 μM, green squares), while at 800 μM, the [CtpS]0.5 value significantly shifted towards polymerization ([CtpS]0.5 = 1.4 μM, blue open diamonds). The maximum kcat values before normalization were 6.7, 3.5, and 1.04 s−1 for experiments using 0, 400, and 800 μM CTP, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.032

Mentions: We compared the sharpness of enzyme inhibition in our novel polymerization-based mechanism to that of previously characterized mechanisms of enzyme inhibition such as competitive and allosteric inhibition (Figure 8D; Supplementary file 1). We found that, among the mechanisms examined, the ones involving polymerization-based negative feedback yield the sharpest decrease in enzyme activity when CTP levels are increased, thereby enabling tight regulation of CTP production by CTP levels. Our estimate based on average CtpS filament length of the value of the nucleation energy yields extremely sharp transitions (see Figure 8D, where this estimate was used, and our discussion of response coefficients in Supplementary file 1). This sharpness is apparent in comparing the concentration dependences of CtpS specific activity in the presence of CTP. The CTPS0.5 value at 400 μM CTP is slightly shifted compared to no CTP. At 800 μM, the CtpS0.5 value is substantially decreased and the curvature more concave (Figure 8—figure supplement 3).


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)

CTP binding enhances polymerization with a sharp response.The concentration required to reduce CtpS-specific activity (kcat) to 50% of its maximum value, [CtpS]0.5, is inversely related to the affinity of the polymer for Ctps tetramers. In the absence of CTP, the [CtpS]0.5 value is 3.3 μM (red circles). At a CTP concentration near the IC50 value (400 μM), the [CtpS]0.5 value is slightly reduced (2.8 μM, green squares), while at 800 μM, the [CtpS]0.5 value significantly shifted towards polymerization ([CtpS]0.5 = 1.4 μM, blue open diamonds). The maximum kcat values before normalization were 6.7, 3.5, and 1.04 s−1 for experiments using 0, 400, and 800 μM CTP, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.032
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig8s3: CTP binding enhances polymerization with a sharp response.The concentration required to reduce CtpS-specific activity (kcat) to 50% of its maximum value, [CtpS]0.5, is inversely related to the affinity of the polymer for Ctps tetramers. In the absence of CTP, the [CtpS]0.5 value is 3.3 μM (red circles). At a CTP concentration near the IC50 value (400 μM), the [CtpS]0.5 value is slightly reduced (2.8 μM, green squares), while at 800 μM, the [CtpS]0.5 value significantly shifted towards polymerization ([CtpS]0.5 = 1.4 μM, blue open diamonds). The maximum kcat values before normalization were 6.7, 3.5, and 1.04 s−1 for experiments using 0, 400, and 800 μM CTP, respectively.DOI:http://dx.doi.org/10.7554/eLife.03638.032
Mentions: We compared the sharpness of enzyme inhibition in our novel polymerization-based mechanism to that of previously characterized mechanisms of enzyme inhibition such as competitive and allosteric inhibition (Figure 8D; Supplementary file 1). We found that, among the mechanisms examined, the ones involving polymerization-based negative feedback yield the sharpest decrease in enzyme activity when CTP levels are increased, thereby enabling tight regulation of CTP production by CTP levels. Our estimate based on average CtpS filament length of the value of the nucleation energy yields extremely sharp transitions (see Figure 8D, where this estimate was used, and our discussion of response coefficients in Supplementary file 1). This sharpness is apparent in comparing the concentration dependences of CtpS specific activity in the presence of CTP. The CTPS0.5 value at 400 μM CTP is slightly shifted compared to no CTP. At 800 μM, the CtpS0.5 value is substantially decreased and the curvature more concave (Figure 8—figure supplement 3).

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