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J Biol Chem, Vol. 274, Issue 40, 28240-28245, October 1, 1999

Functional Linkage between the Glutaminase and Synthetase Domains of Carbamoyl-phosphate Synthetase
ROLE OF SERINE 44 IN CARBAMOYL-PHOSPHATE SYNTHETASE-ASPARTATE CARBAMOYLTRANSFERASE-DIHYDROOROTASE (CAD)

From the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

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 HCO₃, 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 Ser⁴⁴ 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 Ser⁴⁴ GLN domain and the Escherichia coli carbamoyl-phosphate synthetase 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 interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser⁴⁴  Ala mutant than that needed for carbamoyl phosphate synthesis. Ser⁴⁴, 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.

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