Large-scale filament formation inhibits the activity of CTP synthetase.
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.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.Show MeSH
Mentions: Because CtpS filament formation is conserved between divergent organisms, we hypothesized that CtpS polymerization may regulate its conserved enzymatic function. We therefore designed a system to simultaneously monitor the assembly and activity of purified E. coli CtpS. We used a fluorometer to assay CtpS assembly by right-angle light scattering and CtpS activity by the specific absorbance of its CTP product. CtpS assembly and activity were assayed across a range of enzyme concentrations in activity buffer containing saturating amounts of substrates (UTP, ATP, and glutamine) as well as GTP and Mg2+ (referred to as ‘activity buffer’ throughout the text) (Figure 1B). CtpS protein was first pre-incubated in an incomplete activity buffer without glutamine to favor active tetramer formation. CTP production was then initiated by the addition of glutamine to form a complete activity buffer. The formation of well-ordered filaments was confirmed by negative stain electron microscopy (EM) (Figure 1C). Interestingly, at CtpS levels where robust changes in light scattering are observed (above approximately 1–2 μM), CtpS activity (determined by the rate of CTP production per enzyme) sharply decreases (Figure 1B, Figure 1—figure supplements 1 and 2). This abrupt transition in activity state supports the hypothesis that there is a threshold for polymerization and that polymerization is inhibitory. Noise and nonlinearity in the light scattering data make it difficult to determine an exact critical concentration value. However, based on correlation between light scattering and CTP production changes, we predict the assembly threshold of CtpS to be approximately 1–2 μM. The cellular level of CtpS protein in E. coli grown in minimal media was measured at 2.3 μM (Figure 1—figure supplement 3), indicating that the CtpS polymerization observed in vitro may be physiologically favorable.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.