Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.5.5 (CPS)
 1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.
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PMID:Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain. 254 74

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.
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PMID:Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain. 274 25

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.
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PMID:Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase. 282 12