Aquifex aeolicus, an organism that flourishes at 95°C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in k cat with little change in the K m for the substrates, carbamoyl phosphate and aspartate. The K m for both substrates was 30 -40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.Aquifex aeolicus, one of the most hyperthermophilic eubacteria thus far discovered, is classified as a hydrogen-oxidizing, microaerophilic, obligate chemolithoautotroph (1). This marine organism is related to the filamentous bacteria isolated from the hot springs in Yellowstone near the turn of the last century (2, 3). One intriguing question is how unstable metabolites are preserved from thermal degradation in A. aeolicus and other hyperthermophiles. For example, carbamoyl phosphate, a key intermediate in both pyrimidine and arginine biosynthetic pathways, has a half-life of less than 2 s at 100°C and decomposes to toxic cyanate, a promiscuous alkylating agent (4, 5).In the pyrimidine biosynthetic pathway, carbamoyl phosphate is used as a substrate, along with aspartate, for the formation of carbamoyl aspartate in a reaction catalyzed by aspartate transcarbamoylase (ATCase 1 ; EC 2.1.3.2).Carbamoyl phosphate ϩ aspartate 3 carbamoyl aspartate ϩ P i REACTION 1