Determination of ligand binding: partial and full saturation of aspartate transcarbamylase. Applicability of a filter assay to weakly binding ligands.

Abstract

Carbamyl phosphate and succinate each bind to six sites in the hexameric aspartate transcarbamylase from Escherichia coli when both ligands are present in saturating concentrations. Their respective dissociation constants are 2.4 and 1400 muM. Positive homotropic interaction, shown earlier for the association of succinate with the enzyme in the presence of carbamyl phosphate (Changeux, J.-P., Gerhart, J.C., and Schachamn, H.K. (1968) Biochemistry 7, 513-538), is also found for carbamyl phosphate binding in the presence of succinate. Apparent half-of-the-sites saturation, previously described for carbamyl phosphate binding in the absence of succinate (Rosenbusch, J.P., and Griffin, J.H. (1973) J. Biol, Chem. 248, 5063-5066), also occurs when succinate binds to the enzyme in the absence of carbamyl phosphate. A second class of three low affinity sites for carbamyl phosphate could be detected in the enzyme when succinate was absent. These results indicate that aspartate transcarbamylase exists in an asymmetric state under several defined conditions. The results reported were obtained with a highly sensitive filter bonding assay, modified to allow the study of the protein-ligand interactions with dissociation constants in the millimolar range. The assay is described in detail. Its validity is demonstrated by the good correlation of the results obtained with those observed with independent methods.