Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Abstract

SummaryArginase is a binuclear Mn 21 -metalloenzyme of urea cycle that catalyzes the conversion of L-arginine to L-ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co 21 , and has lower catalytic activity. To understand the differences in the biochemical properties as well as activity compared to other arginases, we carried out a detailed investigation of different metal reconstituted H. pylori arginases that includes steady-state kinetics, fluorescence measurement, pH-dependent and oligomerization assays. Unlike other arginases (except human at physiological pH), the Co 21 -and Mn 21 -reconstituted H. pylori enzymes exhibit cooperative mechanism of arginine hydrolysis, and undergo self-association and activation with increasing concentrations. Analytical gel-filtration assays in conjunction with the kinetic data showed that the protein exists as a mixture of monomer and dimer with monomer being the major form (other arginases exclusively exist as a trimer or hexamer) but the dimer is associated with higher catalytic activity. The proportion of dimer is found to decrease with increasing salt concentrations indicating that salt bridges play important roles in dimerization of the protein. Furthermore, the fluorescence measurement showed that Co 21 ions play an important role in the local tertiary structure of the protein than Mn 21 . This is consistent with the pH-dependent studies where the Co 21 -enzyme showed a single ionization compared to the double in the Mn 21 -enzyme. Thus, this study presents the detailed biochemical and spectroscopic investigations into the differences in the biochemical properties and activity between H. pylori and other arginases.

Insight into the role of a unique SSEHA motif in the activity and stability of Helicobacter pylori arginase

“…For wild type, the ratio of the concentration of the apo-protein to the metal was determined, which showed highest activity. The ratio was found to be 20:1 and 500:1 for Co 21 and Mn…”

“…The kinetic assay was performed using a reported procedure (21), where the metal was incubated with the apo-protein. The reactions were carried out by mixing the metal incubated apo-protein with varying concentrations of arginine.…”

“…The concentration of the apo-protein was kept 1-2 lM. The concentration of the metal was used in large excess over the apo-enzyme for both Co 21 -and for Mn 21 -proteins. The mixture was heat activated at 50 8C for about 30 min as reported earlier (4) and then passed through Sephadex G-25 columns, which was equilibrated with 20 mM Tris, pH 7.4.…”

“…We recently reported that a disulfide bond between Cys66 and Cys73 in the H. pylori enzyme is important for the stability rather than the catalysis (20). This enzyme shows several unique features compared to the other arginases such as highest activity with Co 21 as a metal cofactor than Mn 21 , low pH optimum (6.1 instead of 9.5), cooperative mechanism of arginine hydrolysis in the pH range 6.1-9.8, self-association and activation of the protein, and significant sequence differences at the N-and C-terminal sites (4,21). Moreover, the H. pylori enzyme contains all signature motifs except 88 SSEHA 92 (GGDHS in other arginases), although the metal binding His91 residue has been shown to be conserved.…”

“…H. pylori contains the rocF gene coding for arginase (3,4). Arginase (EC 3.5.3.1) is a binuclear Mn 21 -metalloenzyme that catalyzes the conversion of L-arginine to L-ornithine and urea (5). The enzyme is widely present throughout the evolutionary spectrum including bacterium, plant, yeast, and vertebrates (6).…”