Analysis of two purified mutants of Escherichia coli aspartate transcarbamylase with single amino acid substitutions.

Two mutant versions of Escherichia coli aspartate transcarbamylase have been purified and analyzed kinetically. Each of these mutant enzymes contains a single amino acid different from the wild-type enzyme, which was introduced by suppression of a nonsense codon within the E. coli pyrB gene. These enzymes exhibited alterations in both homotropic and heterotropic interactions with little change in specific activity. Depending upon the site of the substitution, the allosteric interactions have been either enhanced or diminished over the wild-type enzyme. Carbamyl phosphate saturation curves indicate that aspartate and carbamyl phosphate homotropic co-operativity are separable. Experiments employing the allosteric effectors indicate that the transmission of the regulatory effect is dependent upon the structure of the catalytic subunit, and that CTP inhibition can be partially decoupled from ATP activation. The kinetics of one of the mutants is unusually sensitive to dissociation at elevated temperatures. This sensitivity may be due to weakened interactions between the subunits of the enzyme.