Modes of modifier action in E. coli aspartate transcarbamylase

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate P_i. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S_0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.