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

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Abstract
Various hybrids of aspartate transcarbamoylase (EC-2.1.3.2) of Escherichia coli were constructed from native regulatory subunits and mixtures of active and inactive (pyridoxylated) catalytic chains in specific arrangements within the 2 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 6, 4, 3, 2 and 1 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 1 contained 2 active sites in 1 catalytic subunit and none in the other, and the 2nd hybrid had 1 active site in each catalytic subunit. These 2 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. There are apparently 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 2-state model of Monod et al.