title: Quaternary constraint in hybrid of aspartate transcarbamylase containing wild-type and mutant catalytic subunits.
abstract: Unusual quaternary constraint in the regulatory enzyme, aspartate transcarbamylase (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, was demonstrated with a hybrid composed of one inactive "catalytic" subunit from a mutant strain and one active catalytic subunit and three regulatory subunits from the wild-type strain. The hybrid had a high affinity for three molecules of the bi-substrat analog, N-(phosphonacetyl)-L-aspartate, compared to the six strong binding sites in the wild-type enzyme and none in the mutant. However, the Vmax of the hybrid was only about 25% that of the wild-type enzyme. In addition, the hybrid exhibited a very low apparent affinity for the substrate, aspartate [Michaelis constant (Km) about 90 mM], as compared to the wild-type enzyme (apparent Km of 7 mM). No homotropic effect was observed for the hybrid in the absence of nucleotides as contrasted to the cooperativity of the wild-type enzyme; also, large changes in the Vmax of the hybrid were caused by the addition of the nucleotide effectors, CTP and ATP, which do not affect the Vmax of the wild-type, but influence only the cooperativity and the apparent Km. Although the hybrid undergoes a ligand-promoted conformational change analogous to that of the wild-type enzyme, this transition required a 20-fold higher concentration of the substrate analog, succinate. It appears that the "paralysis" of the wild-type catalytic subunit in the hybrid can be attributed to subunit interactions which constrain the molecule in a low-affinity state.
mesh_terms: D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006824:Hybridization, Genetic; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001221:Aspartate Carbamoyltransferase