Carbamyl phosphate modifies the T quaternary structure of aspartate transcarbamylase, thereby facilitating the structural transition associated with cooperativity

The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues, thereby providing a direct means to monitor the amount of different quaternary structures present in solution. Scattering curves in the presence of variable concentrations of several such substrate analogues were recorded using an area detector. Data were analyzed by singular-value decomposition without any prior assumption as to the number of quaternary structure states. They can all be accounted for with only two states. The structural transition appears to be concerted, a conclusion whose validity has been extended to higher resolution and to other combinations of ligands than previously studied using a linear detector. The titration curve with the bisubstrate analogue N(phosphonacetyl)-L-aspartate (PALA) alone is identical to that previously obtained, while that in the additional presence of carbamyl phosphate (CP) shows a clear R shift. However, the scattering pattern of the ATCase-CP complex is shown to be different from the T pattern of the unligated enzyme, though not a linear combination of the rand R patterns. Therefore, we conclude that CP acts by modifying the quaternary structure of the unligated enzyme to a T-like structure, a structure more easily converted to the R conformation by subsequent addition of PALA.