Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity highaffinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50 + Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50 + Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steadystate kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50 + Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.Keywords: aspartate carbamoyltransferase; quaternary structure; single amino acid mutation; solution X-ray scattering Aspartate transcarbamoylase (EC 2.1.3.2.) from Escherichia coli catalyzes the first step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form Ncarbamoyl-L-aspartate and phosphate. The reaction mechanism is "preferred order" with carbamoyl phosphate binding before aspartate, and N-carbamoylaspartate leaving before phosphate (Hsuanyu & Wedler, 1987). The E. coli enzyme is endowed with

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure 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-5O/Asp-100 + Ala enzyme is fourfold more active than the Glu-50 -+ Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-IO0 -+ 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-(phosphonacety1)-L-aspartate (PALA). At subsaturating concentrations 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 by 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 succinate only convert a fraction of the Glu-50/Asp-100 -+ Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50 + Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100 + Ala enzyme.Keywords: domain closure; homotropic cooperativity; protein structure-function; site-specific mutagenesis; solution X-ray scattering *We dedicate this paper to the memory of our friend and colleague Escherichia coli aspartate transcarbamoylase (ATCase; EC Frederick C. Wedler, whose work on the catalytic mechanism of aspar-2.1.3.2) catalyzes the committed step in the biosynthesis of pytate transcarbamoylase has been fundamental to the understanding of rimidine nucleotides: the reaction between carbamoyl phosphate this complex enzyme. His personal and intellectual contributions to the scientific community will be sorely missed.and L-aspartate to form N-carbamoyl-L-aspartate and inorganic Reprint requ...