Alteration of the allosteric properties of aspartate transcarbamoylase by pyridoxylation of the catalytic and regulatory subunits

Abstract

Extensive modification of aspartate transcarbamoylase (EC-2.1.3.2) from Escherichia coli with pyridoxal 5''-phosphate followed by reductiion of the Schiff base with sodium borohydride caused only partial inactivation of the enzyme. Under comparable conditions, virtually complete loss of enzyme activity is obtained with the free catalytic subunits. The pyridoxylated, intact enzyme containing more than 60% of the bound puridoxamine phosphate on the regulatory subunits exhibited considerable cooperativity, inhibition by CTP and activation by ATP. When the modification was performed in the presence of the ligands which bind to the catalytic sites, the resulting product had virtually the same activity as the native enzyme, but it exhibited significantly reduced cooperativity and virtually no inhibition by CTP. The pyridoxylation of the regulatory subunits within the intact enzyme was enhanced markedly in the presence of ligands as compared with the reactivity of these subunits when the modification was performed in the absence of the active site ligands. Both types of pyridoxylated derivatives exhibited the ligand-promoted conformational changes characteristic of the native enzyme. Spectrophotometric studies of inactive pyridoxylated catalytic subunits and intact enzyme showed that the substrate (carbamoyl phosphate) bound strongly but that the substrate analogue (succinate) did not bind. Both the pyridoxylation experiments in the presence and absence of ligands and the spectral behavior of a hybrid containing 1 native and 1 pyridoxylated catalytic subunit indicated that ligand binding was accompanied by a conformational change in the intact enzyme molecules.