Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase - Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD)

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate syn thetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctio nal protein that also catalyzes the second and third steps of pyrimidine bi osynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. Th ere is a functional linkage between these domains such that glutamine hydro lysis on the GLN domain does not occur at a significant rate unless ATP and HCO3-, the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenua tion subdomains, In the separately cloned GLN domain, the catalytic subdoma in is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in th e GLN attenuation domain with alanine increases the k(cat)/K-m for glutamin e hydrolysis 680-fold. The formation of a functional hybrid between the mam malian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synt hetase large subunit had little effect on glutamine hydrolysis, In contrast , ATP and HCO3- did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpreta tion, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis we re no longer coordinated. Approximately 3 times more glutamine was hydrolyz ed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate s ynthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis .