Isolation and preliminary characterization of single amino acid substitution mutants of aspartate carbamoyltransferase.
In order to isolate functional Escherichia coli aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC2.1.3.2) with single amino acid replacements, a series of pyrB nonsense mutants has been isolated. These nonsense mutants were induced by 2-aminopurine mutagenesis and selected by a combination of antibiotic treatments, direct enzyme assays, and suppressibility tests. Suppression of the pyrB nonsense mutation with various suppressors, which insert different amino acids, has resulted in the formation of a series of mutant aspartate carbamoyltransferases, each differing in one amino acid from the wild-type enzyme. After partial purification, kinetic studies revealed that some of the mutant enzymes had altered homotropic and heterotropic interactions. The mutants that had a tyrosine insert showed the most pronounced changes, followed by those with a serine insert. The mutants having a glutamine insert, howevr, were indistinguishable from the wild-type enzyme, supporting the conclusion that, because of the specificity of the mutagen, the glutamine insert had regenerated the wild-type enzyme.
D015075:2-Aminopurine / Q000494:pharmacology; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004269:DNA, Bacterial; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic
D004269:DNA, Bacterial; D015075:2-Aminopurine; D003570:Cytidine Triphosphate; D001221:Aspartate Carbamoyltransferase