Analysis of mutant Escherichia coli aspartate transcarbamylases isolated from a series of suppressed pyrB nonsense strains

Abstract

A large collection of mutant versions of Escherichia coli aspartate transcarbamylase (carbamoylphosphate: l-aspartate carbamoyltransferase, EC 2.1.3.2) differing from the wild-type enzyme by a single amino acid has been characterized. These mutant aspartate transcarbamylases were produced by suppression of nonsense codons in the pyrB gene of E. coli K12. By using different suppressors, up to four different enzymes were produced that had their amino acid substitutions at the identical position in the enzyme's primary structure. Thirty-four of these mutant aspartate transcarbamylases derived from nine pyrB nonsense mutations, are described here. These represent enzymes from our collection that exhibit substantial alterations in homotropic and or heterotropic interactions. Suppression of most of the nonsense mutations with supB yields enzymes indistinguishable from the wild-type enzyme, supporting the conclusion that this suppressor has a high probability of regenerating the wild-type enzyme. Enzymes with tyrosine, serine and lysine insertions for the nonsense codon showed significant alterations in kinetic properties. The aspartate concentration at one-half maximal velocity ([S] 0.5) was reduced to 7.0 m m for some mutants and increased to greater than 500 m m for others, compared to 12.7 m mfor the wild-type enzyme. The allosteric interactions of many of the mutants were altered substantially, for example, some of the enzymes exhibited almost hyperbolic kinetics while others exhibited kinetics even more sigmoidal than the wild-type enzyme. ATP activation and CTP inhibition showed both co-ordinated and independent alterations. The changes in the kinetic properties induced by the mutations were reflected in alterations in the kinetic properties of the catalytic subunits derived from the mutant enzymes. However, enhanced substrate affinity for the native enzyme does not always reflect enhanced substrate affinity for the catalytic subunit. The kinetic data derived from these mutant enzymes have revealed correlations between the inserted amino acids and the homotropic and heterotropic interactions of the enzyme.