N-acetylglutamate 5-phosphotransferase of Pseudomonas Aeruginosa. Catalytic and Regulatory Properties

Eur J Biochem. 1975 Mar 17;52(2):377-93.

Abstract

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.

MeSH terms

  • Cations, Divalent
  • Enzyme Activation / drug effects
  • Glutamates
  • Hydrogen-Ion Concentration
  • Kinetics
  • Magnesium / pharmacology
  • Phosphotransferases / metabolism*
  • Pseudomonas aeruginosa / enzymology*
  • Structure-Activity Relationship

Substances

  • Cations, Divalent
  • Glutamates
  • Phosphotransferases
  • Magnesium