Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

Abstract

The cooperativity and feedback inhibition exhibited by the regulatory enzyme, aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase; EC 2.1.3.2) from Escherichia coli was generally attributed to ligand-promoted conformational changes involving alterations in the subunit interactions. However, no quantitative estimates were made of the effect of ligands on the strength of the intersubunit bonding domains. The native enzyme, composed of 2 catalytic trimers crosslinked by 3 regulatory dimers, showed little tendency to dissociate in neutral buffers at room temperature. In addition, very little exchange was observed in 2 h between subunits within the intact enzyme and free subunits. Although exchange was enhanced in solutions of low ionic strength containing the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, the rates of exchange were too small to permit reliable estimates of the weakening of the bonding domains caused by the ligand. Studies were conducted, therefore, on a less stable oligomeric complex which resembled the native enzyme in structure and allosteric behavior but lacked 1 regulatory subunit. These molecules, containing only 4 bonding domains between the catalytic and regulatory polypeptide chains (compared to 6 in the native enzyme), disproportionate to form the more stable native enzyme and free catalytic subunits. An electrophoretic technique was described for measuring the rate of disproportionation which was controlled by the rupture of the intersubunit bonding domains. This rate was enhanced about 300-fold upon the addition of the active-site ligand. Hence the ligand-promoted allosteric conversion of the enzyme-like complex from the constrained to the relaxed conformation involved a substantial weakening of the intersubunit interactions corresponding to about 1.7 kcal/mol (7.1 kJ/mol) per bonding domain between a catalytic and a regulatory chain.