Cooperative interactions in aspartate transcarbamoylase. 1. Hybrids composed of native and chemically inactivated catalytic polypeptide chains.

Abstract

Hybrids of aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate: L-aspartate carbamoyltransferase) from Escherichia coli containing native (active) and pyridoxylated (inactive) catalytic polypeptide chains were constructed by a procedure involving the reversible acylation of amino groups with 3,4,5,6-tetrahydrophthalic anhydride. This technique exploited the charges contributed by the tetrahydrophthaloyl groups as a "chromatographic handle" for separating the various species. Enzyme-like molecules containing one fully active and one inactive catalytic subunit showed cooperative kinetic behavior, considerable inhibition by CTP, and a substantially increased apparent K(m) compared to the native enzyme. Similar properties were observed for an intrasubunit hybrid containing one inactive catalytic polypeptide chain in each subunit. The cooperative inter-and intra-subunit hybrids also exhibited conformational changes similar to those found for the native enzyme upon the addition of stereospecific ligands. These observations, taken together with data for other complexes of catalytic and regulatory subunits, illustrate the importance of the architecture of aspartate transcarbamoylase and the quaternary constraint stemming from the subunit interactions.