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: To determine if polymerization indeed inhibits CtpS activity, we assayed the activity of polymers purified by ultracentrifugation. The polymer-containing pellet was least enzymatically active immediately after centrifugation and CtpS activity increased as the polymers in the pellet disassembled (Figure 1D, Figure 1—figure supplements 4 and 5). CtpS polymers are thus inactive or much less than maximally active and polymerization is readily reversible. We directly demonstrated the reversibility of CtpS assembly and inactivation by first allowing CtpS to polymerize in activity buffer (with all substrates present) and then adding 1 mM UTP and ATP. Upon addition of these substrate nucleotides, we observed a sharp decrease in light scattering that corresponded to a sharp increase in CtpS activity. This transition was followed by a gradual increase in light scattering and corresponding decrease in activity back to the initial residual level (Figure 1E). Control experiments confirmed that the decrease in CtpS polymerization was not due to mechanical disruption by substrate addition (Figure 1—figure supplement 6). The correlation between the decrease in light scattering and the initiation of CTP production at the time of substrate addition indicates that substrate addition leads to rapid depolymerization and subsequent enzyme reactivation. Immediately after this point, we observed an increase in both CTP levels and polymerization. We therefore conclude that polymerized CtpS enzymes are inactive and must disassociate from the polymer to resume normal enzymatic activity. Despite the fact that polymerization occurs in a buffer containing substrates, polymerization only occurs with CTP production, suggesting that polymerization is triggered not by the initial substrates, but rather by the accumulation of CTP product.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.