Wheat-germ aspartate transcarbamoylase: revised purification, stability and re-evaluation of regulatory kinetics in terms of the Monod-Wyman-Changeux model.

A revised and simplified purification scheme for aspartate transcarbamoylase (ATCase) from wheat-germ is reported, with an eightfold increase in scale (yielding approximately 10 mg of the pure protein from 4 kg of wheat-germ), and improved characteristics of stability and regulatory kinetics. The ATCase obtained is greater than 96% pure, as judged by polyacrylamide gel electrophoresis. The long-term stability (i.e. on a time-scale of several hours to weeks) of the activity of the purified enzyme, under various storage conditions, was investigated. At 4 degreesC and pH 7.5, stability was found to be strongly dependent on protein concentration (increased stability at high concentration), buffer concentration (decreased stability at high buffer concentration) and the inclusion of glycerol (increased stability with increasing glycerol concentration). The enzyme is routinely stored at 4 degreesC, in 0. 05 m Tris/HCl buffer containing 25% glycerol and at high protein concentration (approximately 1 mg.mL-1, or 10 microm in trimers). Under these conditions, the half-life of the enzyme activity is greater than 300 days. Over the time-scale of kinetic experiments (up to 20 min), the diluted activity (at around 1 nm of ATCase, in the presence of ligands) is completely stable. The specific activity remains constant in the range 0.1-10 nm, in the absence and presence of ligands, showing that dissociation of the trimeric enzyme into its subunits is negligible. Steady-state kinetics were examined using the enzyme at a concentration of 1.3 nm. Initial-rate curves for both allosteric ligands, carbamoylphosphate (CP) and uridine 5'-monophosphate (UMP), showed pronounced sigmoidicity, each in the presence of the other. In the absence of UMP, initial-rate curves for CP are hyperbolic. The initial rate data fit reasonably well to a trimeric Monod-Wyman-Changeux model, suggesting a two-state conformational mechanism, greatly favouring the active (R) state when both ligands are absent, in which the R-state binds CP exclusively (dissociation constant = 23.2 microm), and the T-state binds UMP exclusively (dissociation constant = 0.49 microm). This regulatory behaviour was found to be quite stable, and was indistinguishable from that of the enzyme in a freshly made crude extract, even after storage of the pure sample for 5 months. This enzyme preparation is therefore free of the anomalous allosteric kinetics produced by a previous purification scheme, in which the affinity for UMP was markedly reduced, CP rate curves showed no sigmoidicity, while UMP rate curves had sigmoidicity exaggerated by a low maximum.