title: Carbamoyl-phosphate synthase in Phycomyces blakesleeanus.
abstract: A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
authors.forename: M J
authors.initials: MJ
authors.lastname: Alonso
authors.forename: D
authors.initials: D
authors.lastname: De Arriaga
authors.forename: J
authors.initials: J
authors.lastname: Soler
mesh_terms: D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009090:Mucorales / Q000201:enzymology*; D010800:Phycomyces / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
chemical_list: D046911:Macromolecular Substances; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)