Concerted allosteric transition in hybrids of aspartate transcarbamoylase containing different arrangements of active and inactive sites.

Various hybrids of aspartate transcarbamoylase of Escherichia coli were constructed from native regulatory subunits and mixtures of active and inactive (pyridoxylated) catalytic chains in specific arrangements within the two catalytic subunits. The kinetic and physical properties of these well-defined hybrids were studied in order to determine the effects of reducing the number of substrate binding sites and distributing the active and inactive chains in different ways. Experiments on enzyme-like molecules containing six, four, three, two, and one active sites showed that the Hill coefficient decreased and the apparent Km increased as the number of active chains in the hybrids was reduced. The maximum inhibition and activation by the nucleotide effectors, CTP and ATP, were independent of the composition of the enzyme-like molecules. Two hybrids were of particular interest since one contained two active sites in one catalytic subunit and none in the other, and the second hybrid had one active site in each catalytic subunit. These two hybrids exhibited identical kinetic behavior despite the markedly different structural arrangements. The ligand-promoted conformational changes of the hybrids monitored both by sedimentation velocity measurements and the reactivity toward p-hydroxymercuribenzoate were similar to those of the native enzyme. These results indicate that there are no discrete "cooperative units" within the enzyme molecules but rather that the allosteric transition promoted by ligands is fully concerted. The various kinetic and physical properties can be accounted for satisfactorily in terms of the two-state model of Monod et al.