GLU-50 IN THE CATALYTIC CHAIN OF ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE PLAYS A CRUCIAL ROLE IN THE STABILITY OF THE R-QUATERNARY STRUCTURE

Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a se t of interdomain bridging interactions between the 2 domains of the ca talytic chain; these interactions are critical for stabilization of th e high-activity high-affinity form of the enzyme. The mutant enzyme wi th an alanine substituted for Glu-50 (Glu-50 --> Ala) exhibits signifi cantly 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-phosphonace tyl-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 scatterin g pattern is hardly altered by a combination of carbamoyl phosphate an d succinate. Addition of ATP under these conditions does result in a s light shift toward the R structure. Stead-state kinetic studies indica te that, in contrast to the wild-type enzyme, the Glu-50 --> Ala enzym e is activated by PALA at saturating concentrations of carbamoyl phosp hate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not und ergo the normal T to R transition upon binding of the physiological su bstrates and verifies the previous suggestion that the interdomain bri dging interactions involving Glu-50 are critical for the creation of t he high-activity, high-affinity R state of the enzyme.