title: The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.
abstract: The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.
authors.affiliation: Instituto de Biomedicina de Valencia (Consejo Superior de Investigaciones Científicas), C/Jaime Roig 11, 46010 Valencia, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Uriarte
authors.forename: A
authors.initials: A
authors.lastname: Marina
authors.forename: S
authors.initials: S
authors.lastname: Ramón-Maiques
authors.forename: I
authors.initials: I
authors.lastname: Fita
authors.forename: V
authors.initials: V
authors.lastname: Rubio
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D002219:Carbamates / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D003001:Cloning, Molecular; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D013293:Enterococcus faecalis / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D008958:Models, Molecular; D009952:Ornithine / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D014162:Transfection
chemical_list: D002219:Carbamates; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)