title: The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate.
abstract: The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, Wayne State University, Detroit, Michigan 48201.
authors.forename: M G
authors.initials: MG
authors.lastname: Chaparian
authors.forename: D R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D000878:Anthranilate Synthase; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism*; D002384:Catalysis; D006224:Cricetinae; D004080:Dihydroorotase / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D005973:Glutamine / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008647:Mesocricetus; D009097:Multienzyme Complexes / Q000378:metabolism*; D019884:Nitrogenous Group Transferases; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
chemical_list: C033626:CAD trifunctional enzyme; D004952:Esters; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D005973:Glutamine; D002221:Carbamyl Phosphate; D000641:Ammonia; D014166:Transferases; D001221:Aspartate Carbamoyltransferase; D019884:Nitrogenous Group Transferases; D004080:Dihydroorotase; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)