In situ characterization of Helicobacter pylori arginase.
The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.
Mendz
G L
GL
School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, NSW 2052, Australia. g.mendz@unsw.edu.au
Holmes
E M
EM
Ferrero
R L
RL
D001119:Arginase / Q000737:chemistry*; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001692:Biological Transport; D002413:Cations, Divalent / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016480:Helicobacter pylori / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009952:Ornithine / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D001120:Arginine; D009952:Ornithine; D001119:Arginase