Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor.

The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate. Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1. The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction [Tsuruta, H., et al. (1990) FEBS Lett. 263, 66-68]. After equilibrium had been established, the final scattering pattern was recorded. When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA). This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb [Gouaux, J.E., & Lipscomb, W.N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4205-4208]. Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states. This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)