Physiological evaluation of the role of aspartate transcarbamoylase in the pyrimidine pathway

Abstract

Aspartate transcarbamoylase catalyses the first uniquely committed step of the de novo pyrimidine biosynthesis in Escherichia coli, the condensation of carbamoyl phosphate with aspartate. The genetic expression of the protein is regulated over a physiological range of about twenty fold, and several hundred fold in starvation for pyrimidine. The activity of an existent pool of enzyme is then subjected to multiple metabolic controls. The enzyme exhibits positive cooperativity toward the binding of its substrare aspartate. It is also subject to heterotropic regulation, activated by ATP, inhibited by CTP, and UTP in the presence of CTP. Because of its position at a metabolic branch point which controls a relatively unstable, energetically expensive substrate and its multiple regulation points, ATCase is often seen as a paradigm of a regulatory enzyme. Nevertheless, the ATCases from other enteric bacteria have shown important allosteric variations in the scheme of regulation of ATCase. Furthermore, the heterologous expression of those enzymes heterologous in pyrB' E. coli has revealed an apparent indifference toward the type of ATCase. This study evaluates the influence of the quantity of enzyme and the nature of the regulatory controls of ATCase on the maintenance of the metabolic end-products of the pathway, CTP and UTP. The influence of different ATCases on the triphosphate nucleotide pools and the expression of the other enzymes of the pathway were first determined during homeostatic logarithmic growth. The dynamic response of the triphosphate nucleotide pools to a shift-down of uracil was used to define how the recruitment of the de novo pathway to provide pyrimidines nucleotides was affected the various ATCase enzymes. Finally, the selective advantage of a particular ATCase in maintaining pools of nucleotides was determined.