Abstract
Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. 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.
Highlights
Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that catalyzes the second and third steps of pyrimidine biosynthesis
There is a functional linkage between these domains such that glutamine hydrolysis 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
Carbamoyl phosphate synthesis involves the concerted action of two domains that must act in synchrony
Summary
Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that catalyzes the second and third steps of pyrimidine biosynthesis. The formation of a functional hybrid between the mammalian Ser GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. ATP and HCO3؊ did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Mammalian carbamoyl-phosphate synthetase is part of a large multifunctional protein called CAD [3,4,5], which has aspartate transcarbamoylase and dihydroorotase activities, enzymes that catalyze the second and third steps of the de novo pathway, respectively. The reaction proceeds through a thioester intermediate, and there is evidence that a catalytic subunit of carbamoyl-phosphate synthetase; GLN, the amidotransferase or glutaminase domain or subunit of carbamoyl-phosphate synthetase; GLNase, glutaminase activity; GLN-CPS, the hybrid CPSase consisting of the mammalian GLN domain and the E. coli CPS domain; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; OPA, o-phthaldialdehyde
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