Publications

• Purification of carbamyl phosphate synthetase from frog liver.

  MARSHALL M., METZENBERG R.L., COHEN P.P.

  J Biol Chem 233:102-105(1958) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: effects of CPS1 mutations that concentrate in a central domain of unknown function.

  Diez-Fernandez C., Hu L., Cervera J., Haeberle J., Rubio V.

  Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequ ... >> More

  Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased V(max). Substantial although not dramatic increases in K(m) values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations. Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of "Integrating Domain" for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions. << Less

  Mol. Genet. Metab. 112:123-132(2014) [PubMed] [EuropePMC]

• The pathway of carbonate in the biosynthesis of carbamyl phosphate.

  JONES M.E., SPECTOR L.

  J Biol Chem 235:2897-2901(1960) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Physical and Kinetic Properties of Carbamyl Phosphate Synthetase from Frog Liver.

  Marshall M., Metzenberg R.L., Cohen P.P.

  J Biol Chem 236:2229-2237(1961) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• A KINETIC STUDY OF CARBAMYL PHOSPHATE SYNTHETASE.

  FAHIEN L.A., COHEN P.P.

  J Biol Chem 239:1925-1934(1964) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Molecular characterization of carbamoyl-phosphate synthetase (CPS1) deficiency using human recombinant CPS1 as a key tool.

  Diez-Fernandez C., Martinez A.I., Pekkala S., Barcelona B., Perez-Arellano I., Guadalajara A.M., Summar M., Cervera J., Rubio V.

  The urea cycle disease carbamoyl-phosphate synthetase deficiency (CPS1D) has been associated with many mutations in the CPS1 gene [Häberle et al., 2011. Hum Mutat 32:579-589]. The disease-causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a sy ... >> More

  The urea cycle disease carbamoyl-phosphate synthetase deficiency (CPS1D) has been associated with many mutations in the CPS1 gene [Häberle et al., 2011. Hum Mutat 32:579-589]. The disease-causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a system for recombinant expression, mutagenesis, and purification of human carbamoyl-phosphate synthetase 1 (CPS1), a very large, complex, and fastidious enzyme. The kinetic and molecular properties of recombinant CPS1 are essentially the same as for natural human CPS1. Glycerol partially replaces the essential activator N-acetyl-l-glutamate (NAG), opening possibilities for treating CPS1D due to NAG site defects. The value of our expression system for elucidating the effects of mutations is demonstrated with eight clinical CPS1 mutations. Five of these mutations decreased enzyme stability, two mutations drastically hampered catalysis, and one vastly impaired NAG activation. In contrast, the polymorphisms p.Thr344Ala and p.Gly1376Ser had no detectable effects. Site-limited proteolysis proved the correctness of the working model for the human CPS1 domain architecture generally used for rationalizing the mutations effects. NAG and its analogue and orphan drug N-carbamoyl-l-glutamate, protected human CPS1 against proteolytic and thermal inactivation in the presence of MgATP, raising hopes of treating CPS1D by chemical chaperoning with N-carbamoyl-l-glutamate. << Less

  Hum. Mutat. 34:1149-1159(2013) [PubMed] [EuropePMC]

• Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from human liver.

  Pierson D.L., Brien J.M.

  Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange ch ... >> More

  Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange chromatograhy and electrofocusing The native molecular weight of the enzyme was determined by gel filtration to be 190,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a monomeric molecular weight of 165,000. The isoelectric point of the purified enzyme was 6.05, and only one species of active enzyme was observed during electrofocusing of both purified enzyme preparations and crude liver homogenates. The enzyme exhibited a pH optimum of 7.8. The apparent Michaelis constants for NH4+, HCO3-, MgATP, and the activator, N-acetyl-L-glutamic acid, were 0.8, 6.7, 1.1, and 0.1 mM, respectively. << Less

  J Biol Chem 255:7891-7895(1980) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.