title: Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.
abstract: A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.
mesh_terms: D007700:Kinetics; D008967:Molecular Biology; D009154:Mutation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
chemical_list: D011392:Proline; D010770:Phosphotransferases