National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Biochem J. 2019 Dec 12;476(23):3595-3614. doi: 10.1042/BCJ20190545.
Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (-122HTAYDSDSKHIHG134-) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer - an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.
精氨酸酶是一种双金属酶，主要利用 Mn2+或 Co2+进行催化功能。在人类同源物中，用 Co2+替代 Mn2+会显著降低 Km 值，而不影响 kcat。然而，在幽门螺杆菌对应物（对发病机制很重要）中，无论是在重组全酶中还是在从用 Co2+或 Mn2+生长的 H. pylori 中分离的精氨酸酶中，用 Co2+离子都会使 kcat 增加近 4 倍。这表明这两种同源物的精氨酸酶的活性部位受到这两种金属离子的不同调节。为了研究金属诱导的幽门螺杆菌酶催化活性差异的潜在机制，我们使用了生化、生物物理和微秒分子动力学模拟研究。研究表明，Co2+和 Mn2+离子与蛋白质的结合亲和力的差异与一个包含保守催化 His133 的环（-122HTAYDSDSKHIHG134-）的不同定位有关。因此，His133 和保守的 Glu281 的位置会发生变化。我们发现，Glu281-His133 相互作用对于催化功能至关重要，这在其他同源物中尚未被探索。我们认为，这两个残基之间的接近差异改变了 His133 通过其 pKa 的变化而质子化的比例。这会影响质子转移的效率——精氨酸酶催化 l-精氨酸水解反应的一个关键步骤，从而影响活性。与人类精氨酸酶不同，在幽门螺杆菌同源物中观察到的上述片段的灵活性表明，该区域可能在幽门螺杆菌酶中被探索以设计其特异性抑制剂。
