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: Purified CtpS protein was incubated at 37°C for 20 min in 50 mM Tris HCl (pH 7.8), 10 mM MgCl2, 1 mM UTP, 1 mM ATP, and 0.2 mM GTP to allow tetramer formation. CTP production was initiated by the addition of 10 mM glutamine to create a full activity buffer (referred to in text at ‘activity buffer’) (Ingerson-Mahar et al., 2010) immediately prior to recording of sample measurements. Time between glutamine addition and initiation of sample recording averaged 5 s was based on the amount of time required to load the sample. Reaction was monitored at 37°C for 5 min in a QuantaMaster 40 Fluorometer (Photon Technology International, Birmingham, NJ) equipped with photo multiplier tubes for both scattering and transmittance. Right angle light scattering at 405 nm with a 1 mM slit width detected polymerization, and transmittance at 291 nm with a 0.25-mM slit width detected CTP production with both values reported in arbitrary units. Reactions were performed in 150 μl samples. Polymerization was monitored for 3 min unless otherwise noted. Detection of light scattering and transmittance alternated with an integration time of 1 s. CTP production velocity (kcat, μmol/s) was determined for the first 30 s of the reaction. CTP production was normalized by the concentration of CtpS enzyme in each sample. Due to the fluorometer assay's use of transmittance and a photon multiplier, we compared data collected to data collected over the same concentration range on a more traditional spectrophotometer setup in the Baldwin lab. Comparison yielded the presence of a scaling factor to be applied to the fluorometer data set to yield kcat ranges consistent with published data. Data were scaled to yield the same maximal kcat value for both data sets. The fold-change in activity over the concentrations was similar between the data sets. Overlay of the data are shown in Figure 1—figure supplement 7. Quantification of polymerization was calculated using the difference between the average initial and final values of light scattering for each sample (n = 5 for average) in Figures 1B and 2A and Figure 1—figure supplement 1, Figure 2—figure supplements 1 and 2, and Figure 7—figure supplement 1. All other light scattering values are the actual values of light scattering recorded (in arbitrary units), except where noted in the figure legends.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.