The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold‐adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B‐factors of the R336L crystal structure to that of the wild‐type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half‐of‐sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive).

中文翻译：

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冷活性弧菌碱性磷酸酶的高催化速率需要一个涉及大界面环的氢键网络

在酶的冷适应研究中，表面环在通过残基网络介导通信中的作用仍然是一个相对知之甚少的部分，特别是在它们的四元相互作用方面。来自嗜冷海洋细菌Vibrio splendidus的碱性磷酸酶 (AP)(VAP) 的特点是每个单体中都有一个类似的大表面环，称为大环，它悬停在另一个单体的活性位点上。它可能在 VAP 的高催化效率中起作用，同时伴随着其极低的热稳定性。在这里，我们设计了几种不同的 VAP 变体，目的是消除二聚体界面处的亚基间相互作用。特别是从一个残基 (Arg336) 断开亚基间接触会降低催化有效构象的温度稳定性，并导致催化速率下降 40%。来自冷适应生物的酶的高催化率通常与增加的动态灵活性有关。R336L 晶体结构的相对 B 因子与野生型的比较证实了表面柔韧性在相反单体的环中增加，但在大环中没有。柔韧性的增加导致催化速率降低。大环通过其接触增加了亚基之间的界面面积，并且可能通过更强的联系促进半位点反应机制所需的交替结构循环，因为二聚体在高亲和力（活性）或低磷酰基之间振荡亲和力（非活动）。