title: Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent.
abstract: The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000032:analysis / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents / Q000494:pharmacology; D011487:Protein Conformation
chemical_list: D007202:Indicators and Reagents; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase