Kinetics of aspartate transcarbamylase from Escherichia coli for the reverse direction of reaction.

The reverse reaction of aspartate transcarbamylase in which phosphate or arsenate is first coupled to carbamyl aspartate, followed by elimination of aspartate, has been studied under conditions in which one product, aspartate, is removed. Aspartate is converted to oxalacetate by glutamate-oxalacetate transaminase, and the resulting oxalacetate is converted to malate by the NADH, NAD+ oxidoreductase enzyme malate dehydrogenase. Phosphate and carbamyl aspartate saturation curves are nonsigmoidal. The transition state analogue, N-phosphonacetyl-L-aspartate, activates this reverse reaction substantially. Reverse kinetic parameters of the Haldane type are characteristic of the T-state and correlated with the parameters of the usual forward reaction of the T-state. Phosphate and carbamyl aspartate do not alter the thiol reactivity or sedimentation coefficient of the enzyme. These five results indicate that, under the conditions of these experiments, the reverse reaction does not cause the allosteric transition. In a new assay for the forward reaction we couple phosphate production with NADP reduction using phosphorylase a, phosphoglucomutase, and glucose-6-phosphate dehydrogenase.