TIME-RESOLVED SOLUTION SCATTERING STUDIES ON A MUTANT VERSION OF E. coli ASPARTATE TRANSCARBAMOYLAE. H. Tsuruta, D.P. Baker,^* T.T. Tibbitts,^* S.C. Pastra-Landis,^*+ P. Vachette,^{[[paragraph]]} E. R. Kantrowitz^*. SSRL, Stanford Univ.; Dept. Chemistry, Boston College^*; Dept. Chem. Wheaton College⁺; LURE, Université Paris-sud^{[[paragraph]]}.

E. coli aspartate transcarbamoylase (ATCase) undergoes a large quaternary structure change in the allosteric transition upon binding of various combinations of substrate analogues and natural substrates. Glu-50 near the carbamoyl phosphate binding site is involved in the interdomain interaction that is essential in transforming the enzyme from T state to R state. The Glu-50 -> Ala (E50A) mutation results in an enzyme with low activity, low aspartate affinity and little cooperativity. Enzyme kinetics studies have shown that E50A ATCase may be in an intermediary structure during the enzyme catalysis and that the allosteric effector ATP or the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) can activate the mutant fully to R state. Indeed the three-dimensional structure of the E50A ATCase co-crystallized with PALA is found to be in fully R state. We have carried out time-resolved solution x-ray scattering studies of E50A ATCase during the enzyme reaction with the natural substrates carbamoyl phosphate and aspartate, aimed at characterizing the possible intermediate structure which is short-lived and difficult to study by x-ray crystallography.

A concentrated enzyme solution was mixed with a substrate solution within a stopped-flow rapid mixer, and collection of time-resolved solution scattering curves in the range of 18-130 Å in Bragg spacing immediately followed, using a synchrotron radiation beam. The subsidiary scattering peak recorded during the enzyme reaction was intermediary between the curves recorded for the complete T and R states. The additional presence of ATP converts the curve all the way to that of R structure while at low PALA concentrations there is a lag phase in which the peak stays intermediary for a while then evolves to the curve of the full R structure. Along with the biochemical results, these observations demonstrate the existence of a quaternary structure intermediary between T and R, rather than the coexistence of T and R structures.