National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
IUBMB Life. 2010 Dec;62(12):906-15. doi: 10.1002/iub.401.
Arginase is a binuclear Mn(2+)-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(2+), 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(2+)- and Mn(2+)-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(2+) ions play an important role in the local tertiary structure of the protein than Mn(2+). This is consistent with the pH-dependent studies where the Co(2+)-enzyme showed a single ionization compared to the double in the Mn(2+)-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.
精氨酸酶是尿素循环中的一种双核 Mn(2+)-金属酶，可催化 L-精氨酸转化为 L-鸟氨酸和尿素。与其他精氨酸酶不同，幽门螺杆菌酶对 Co(2+)具有选择性，且催化活性较低。为了了解其生化特性和活性与其他精氨酸酶的差异，我们对不同金属重组的幽门螺杆菌精氨酸酶进行了详细研究，包括稳态动力学、荧光测量、pH 依赖性和寡聚化测定。与其他精氨酸酶（生理 pH 下的人除外）不同，Co(2+)和 Mn(2+)重组的幽门螺杆菌酶表现出精氨酸水解的协同机制，并随着浓度的增加发生自组装和激活。分析凝胶过滤测定与动力学数据表明，该蛋白以单体和二聚体的混合物形式存在，其中单体为主要形式（其他精氨酸酶仅以三聚体或六聚体形式存在），但二聚体与更高的催化活性相关。随着盐浓度的增加，二聚体的比例降低，表明盐桥在蛋白二聚体化中起着重要作用。此外，荧光测量表明 Co(2+)离子在蛋白的局部三级结构中比 Mn(2+)发挥更重要的作用。这与 pH 依赖性研究一致，其中 Co(2+)-酶表现出单离子化，而 Mn(2+)-酶则表现出双离子化。因此，本研究对幽门螺杆菌和其他精氨酸酶之间生化特性和活性的差异进行了详细的生化和光谱学研究。
