N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Purification and ligand-directed association-dissociation.

N-Acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8), the second enzyme of arginine biosynthesis, was purified over 2000-fold from Pseudomonas aeruginosa. The purification procedure involved a heat treatment, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, Sephadex G-150, and hydroxyapatite. The purified enzyme was greater than 90% pure as judged by analytical polyacrylamide gel electrophoresis. A molecular weight of approximately 230000 was obtained by gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 29000. Due to the capacity for self-association, the enzyme can exist in different states of aggregation depending on the nature of ligands and the concentrations of phosphate buffer. As estimated by gel filtration, the molecular weight was about 230000 in the presence of N-acetyl-L-glutamate. With L-arginine, the feedback inhibitor, and MgATP forms of smaller molecular weight (minimum of approximately 65000) were found. A concurrent change in the sedimentation coefficient as a function of ligands was demonstrated by sucrose gradient centrifugation. The synthesis of N-acetylglutamate 5-phosphotransferase was not repressed by exogenous L-arginine or its precursors.