Purification and characterization of a mutant aspartate transcarbamoylase lacking enzyme activity.

A mutant form of aspartate transcarbamoylase (ATCase) from Escherichiu coli which lacks catalytic activity has been purified from a strain which produces large quantities of the inactive protein. Analysis of hybrid enzymes composed of subunits from the mutant and wild type enzyme showed that the mutation was in the structural gene for the catalytic subunits. No alteration of the regulatory subunits was detected. The mutant enzyme had the same sedimentation coefficient, 11.7 S, as the wild type enzyme, and the molecular weights of the individual catalytic and regulatory polypeptide chains of the mutant enzyme evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were the same as the wild type enzyme. Amino acid analysis of the catalytic and regulatory subunits showed no differences, within 3% error, between the two forms of the enzyme. Electrophoresis in polyacrylamide gels containing 8 M urea indicated that the catalytic polypeptide chains of the mutant had a charge difference of -1 relative to those of the wild type enzyme. Ligand binding studies were performed by ultraviolet difference spectroscopy. Although the mutant catalytic subunit bound the substrate, carbamoylphosphate, the dissociation constant, 150 pM, was much greater than that for the wild type catalytic subunit, 9 pM. Neither the substrate analog, succinate, nor the bisubstrate analog, N-(phosphonacetyl)+aspartate (PALA), was bound by the mutant protein, whereas both of these ligands are bound with high affinity by the wild type enzyme. Preparations of the mutant enzyme do not contain a regulatory subunit-deficient species which is usually found in preparations of the wild type enzyme. Moreover, this species could not be generated by limited degradation of the mutant ATCase by mercurials nor by reconstitution of mutant enzyme from mixtures of catalytic and regulatory subunits with the former in excess. These results indicate that the