On conformational changes in the regulatory enzyme aspartate transcarbamoylase.

Our preliminary studies provide clear answers to only some of the basic questions posed above regarding the nature of conformational changes in ATCase. By exploiting the reverse reaction to develop a kinetic active site titration technique we demonstrated that symmetry-related regions of ATCase are affected equally in the allosteric transition even though the bisubstrate analog is bound to only one of the six active sites. This is a vivid illustration of a "global" change in an enzyme, and it provides powerful support for the view that active site ligands promote a concerted transition of the enzyme from the low-activity to the high-activity conformation. Kinetic experiments show that this conformational change is rapid, requiring only tenths of a second. In contrast, the studies with isolated catalytic subunits provide evidence for a much more local conformational change which occurs after the ligand is bound and which is much slower, lasting over a time period of many seconds. Although the experiments on the holoenzyme do not show directly how many atoms are involved in the conformational change or how large are the displacements, they do indicate, especially in conjunction with other studies, that many amino acid residues in both the catalytic and regulatory chains must be implicated in the T----R allosteric transition. Why PALA has such a different local effect on the catalytic subunit than does the combination of carbamoylphosphate and succinate is not clear, but this uncertainty may be resolved by further NMR studies. Determining how the local changes at the active sites are linked to the global transition affecting the quaternary structure of the enzyme remains a formidable problem. Also further work is needed in order to determine whether unliganded ATCase molecules exist in an equilibrium mixture of T and R conformations even prior to the ligand binding event.