Involvement of tryptophan 209 in the allosteric interactions of Escherichia coli aspartate transcarbamylase using single amino acid substitution mutants.
Five mutant versions of aspartate transcarbamylase have been isolated, all with single amino acid substitutions in the catalytic chain of the enzyme. A previously isolated pyrB nonsense mutant was suppressed with supB, supC, supD and supG to create enzymes with glutamine, tyrosine, serine or lysine, respectively, inserted at the position of the nonsense codon. Each of these enzymes was purified to homogeneity and kinetically characterized. The approximate location of the substitution was determined by using tryptic fingerprints of the wild-type enzyme and the enzyme obtained with a tyrosine residue inserted at the position of the nonsense codon. By first cloning the pyrBI operon, from the original pyrB nonsense strain, followed by sequencing of the appropriate portion of the gene, the exact location of the mutation was determined to be at position 209 of the catalytic chain. Site-directed mutagenesis was used to generate versions of aspartate transcarbamylase with tyrosine and glutamic acid at this position. The Tyr209 enzyme is identical with that obtained by suppression of the original nonsense mutation with supC. The two enzymes produced by site-directed mutagenesis were purified using a newly created overproducing strain. Kinetic analysis revealed that each mutant has an altered affinity for aspartate, as judged by variations in the substrate concentration at one-half maximal activity. In addition, the mutants exhibit altered Hill coefficients and maximal activities. In the wild-type enzyme, position 209 is a tryptophan residue that is involved in the stabilization of a bend in the molecule near the subunit interface region. The alteration in homotropic cooperativity seems to be due to changes induced in this bend in the molecule, which stabilizes alternate conformational states of the enzyme.
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013489:Suppression, Genetic; D014364:Tryptophan
D000596:Amino Acids; D014364:Tryptophan; D001221:Aspartate Carbamoyltransferase