Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi. Insights into cooperative and allosteric mechanisms.

Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature, the transition between T- and R-states involves significant changes in the quaternary structure, which were detected by analytical ultracentrifugation. The enzyme is allosterically regulated by ATP (activator) and by CTP and UTP (inhibitors). Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas ATP and UTP act at millimolar concentrations, CTP inhibits at micromolar concentrations, suggesting that in P. abyssi, inhibition by CTP is the major control of enzyme activity. While V(max) increased with temperature, cooperative and allosteric effects were little or not affected, showing that molecular adaptation to high temperature allows the flexibility required to form the appropriate networks of interactions. In contrast to the same enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the carbamyl phosphate analogue phosphonacetate; this difference supports the idea that in native cells ATCase interacts with carbamyl phosphate synthetase to channel the highly thermolabile carbamyl phosphate.