Apparent cooperativity for carbamoylphosphate in Escherichia coli aspartate transcarbamoylase only reflects cooperativity for aspartate.
The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low-affinity T state to a high-affinity R state. In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate. It is shown here that cooperativity for this substrate is only apparent. Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state. Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T-->R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate. Similar results were obtained in the course of continuous-flow-dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state. Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.
England
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P
Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, URA CNRS 1682, Université Pierre et Marie Curie, Paris, France.
Leconte
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C
Tauc
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Hervé
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G
D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002221:Carbamyl Phosphate / Q000737:chemistry / Q000378:metabolism*; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D013379:Substrate Specificity; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid
D013386:Succinates; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase