WEAKENING OF THE INTERFACE BETWEEN ADJACENT CATALYTIC CHAINS PROMOTESDOMAIN CLOSURE IN ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enz yme consisting of two trimeric catalytic subunits and three dimeric re gulatory subunits. Asp-100, from one catalytic chain, is involved in s tabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has b een shown previously to result in increases in the maximal specific ac tivity, homotropic cooperativity, and the affinity for aspartate (Bake r DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to d etermine whether these properties were due to promotion of domain clos ure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp- 100 --> Ala mutation was introduced into the Glu-50 --> Ala holoenzyme , a mutant in which domain closure is impaired. The Glu-50/Asp-100 --> Ala enzyme is fourfold more active than the Glu-50 --> Ala enzyme, an d exhibits significant restoration of homotropic cooperativity with re spect to aspartate. In addition, the Asp-100 --> Ala mutation restores the ability of the Glu-50 --> Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentration s of aspartate, the Glu-50/Asp-100 --> Ala enzyme is activated more by ATP than the Glu-50 --> Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50 --> Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100 --> Ala enzyme is activated b y ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100 --> Ala enzyme by solution X -ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and su ccinate only convert a fraction of the Glu-50/Asp-100 --> Ala enzyme p opulation to the R quaternary structure, a behavior intermediate betwe en that observed for the Glu-50 --> Ala and wild-type enzymes. Solutio n X-ray scattering was also used to investigate the structural consequ ences of nucleotide binding to the Glu-50/Asp-100 --> Ala enzyme.