title: The Study of Carbamoyl Phosphate Synthetase 1 Deficiency Sheds Light on the Mechanism for Switching On/Off the Urea Cycle.
abstract: Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is an inborn error of the urea cycle having autosomal (2q34) recessive inheritance that can cause hyperammonemia and neonatal death or mental retardation. We analyzed the effects on CPS1 activity, kinetic parameters and enzyme stability of missense mutations reported in patients with CPS1 deficiency that map in the 20-kDa C-terminal domain of the enzyme. This domain turns on or off the enzyme depending on whether the essential allosteric activator of CPS1, N-acetyl-L-glutamate (NAG), is bound or is not bound to it. To carry out the present studies, we exploited a novel system that allows the expression in vitro and the purification of human CPS1, thus permitting site-directed mutagenesis. These studies have clarified disease causation by individual mutations, identifying functionally important residues, and revealing that a number of mutations decrease the affinity of the enzyme for NAG. Patients with NAG affinity-decreasing mutations might benefit from NAG site saturation therapy with N-carbamyl-L-glutamate (a registered drug, the analog of NAG). Our results, together with additional present and prior site-directed mutagenesis data for other residues mapping in this domain, suggest an NAG-triggered conformational change in the β4-α4 loop of the C-terminal domain of this enzyme. This change might be an early event in the NAG activation process. Molecular dynamics simulations that were restrained according to the observed effects of the mutations are consistent with this hypothesis, providing further backing for this structurally plausible signaling mechanism by which NAG could trigger urea cycle activation via CPS1.
authors.affiliation: Institute for Biomedicine of Valencia of the Spanish Research Council, Valencia 46010, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Díez-Fernández
authors.affiliation: Medical School of the Catholic University of Valencia, Valencia 46001, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Gallego
authors.affiliation: University Children's Hospital Zurich and Children's Research Center, Zurich 8032, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Häberle
authors.affiliation: Institute for Biomedicine of Valencia of the Spanish Research Council, Valencia 46010, Spain; Centre for Biomedical Networking Research on Rare Diseases of the Carlos III Health Institute, Valencia 46010, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Cervera
authors.affiliation: Institute for Biomedicine of Valencia of the Spanish Research Council, Valencia 46010, Spain; Centre for Biomedical Networking Research on Rare Diseases of the Carlos III Health Institute, Valencia 46010, Spain. Electronic address: rubio@ibv.csic.es.
authors.forename: Vicente
authors.initials: V
authors.lastname: Rubio
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020165:Carbamoyl-Phosphate Synthase I Deficiency Disease / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004795:Enzyme Stability; D005971:Glutamates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014508:Urea / Q000378:metabolism*
chemical_list: D005971:Glutamates; D000641:Ammonia; D014508:Urea; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid
keywords: Allosteric regulation; Carbamoyl phosphate synthetase 1; Enzyme; Hyperammonemia; Inborn errors; Restrained molecular dynamics; Site-directed mutagenesis; Urea cycle diseases