The three-dimensional structure of Escherichia coli aspartate transcarbamoylase complexed with the allosteric effector CTP, shows an interaction between the hydroxyl of Thr-82 in the regulatory chain (Thr-82r) with the gamma-phosphate of CTP (R.P. Kosman, J.E. Gouaux, W.N. Lipscomb, Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for aspartate transcarbamoylase mutants and the mechanism of negative cooperativity, Proteins Struct. Funct. Genet. 15 (1993) 147-176). In order to determine whether the Thr-82r interaction with the gamma-phosphate of CTP is important for either binding of the nucleotide effectors or their function, site-specific mutagenesis was employed. The mutant enzyme in which Thr-82r was replaced by Ala had almost the identical maximal observed specific activity as the wild-type enzyme; however, the mutant enzyme had a significantly increased [Asp]0.5, the aspartate concentration at one-half the maximal observed specific activity, as well as slightly increased homotropic cooperativity. The mutant enzyme was also activated more by ATP and inhibited less by CTP as compared to the wild-type enzyme. In addition, the nucleotide concentration required for one-half maximal effect was increased approx. 3-fold as compared to the corresponding values for the wild-type enzyme. The maximal inhibition of the mutant enzyme, in the presence of UTP and CTP was similar to that observed for the wild-type enzyme; however, higher concentrations of the nucleotides were required to achieve this level of inhibition. The reduced affinity of CTP, UTP and ATP induced by the mutation indicates that the hydrogen bonding interaction between the gamma-phosphate of the nucleotide effector and the side-chain hydroxyl of Thr-82r is important for the binding of the nucleotide effectors to the allosteric site. Furthermore, this interaction is important for the discrimination between CTP and CDP. Finally, the greater homotropic cooperativity, greater [Asp]0.5, diminished CTP inhibition and greater ATP activation of the mutant enzyme correlates with the X-ray structure of the mutant enzyme which shows that the unligated enzyme is in an 'extreme' T-state. These findings add support to the theory that the global stabilization of the enzyme is critical for both the homotropic and heterotropic properties of aspartate transcarbamoylase.