Stines-Chaumeil Claire, Mavré François, Kauffmann Brice, Mano Nicolas, Limoges Benoît
CNRS, Université de Bordeaux, CRPP, UMR 5031, 115 Avenue Schweitzer, F-33600 Pessac, France.
Université de Paris, Laboratoire d'Electrochimie Moléculaire, UMR 7591, CNRS, F-75013 Paris, France.
ACS Omega. 2020 Jan 23;5(4):2015-2026. doi: 10.1021/acsomega.9b04034. eCollection 2020 Feb 4.
The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e., pyrroloquinoline quinone (PQQ) and Ca, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated with two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamics and also to demonstrate that when PQQ first binds to apo-sGDH, it strongly impedes the access of Ca to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca binding loop through the specific mutation of the calcium-coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen-bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB: 5MIN).
通过酶的自发重组来开启其活性的能力，已被证明是酶结构/反应性关系基础研究或生物电子学、合成生物学或生物分析应用中人工信号转导系统设计的一种有价值的工具。特别是，基于来自[具体来源未提及]的载脂蛋白-PQQ依赖性可溶性葡萄糖脱氢酶（sGDH）的自发重组/激活的那些系统得到了广泛发展。然而，sGDH与其两种辅因子，即吡咯并喹啉醌（PQQ）和钙的重组机制仍然未知。这里的目标是通过单周转条件下的停流动力学来阐明这一机制。sGDH的重组表现出双相动力学，这是与两种激活途径相关的方形反应方案的特征。通过完整的动力学分析，我们能够完全预测重组动力学，并且还证明当PQQ首先与脱辅基-sGDH结合时，它会强烈阻碍钙进入其在酶结合位点底部的封闭位置，从而大大减慢sGDH的重组速率。通过对钙配位的P248脯氨酸残基进行特定突变，为钙结合环提供更多灵活性，从而降低钙离子插入的动力学障碍，这种缓慢的钙插入可能会有意加快。重组过程的动态性质还得到了以下观察结果的支持：在脱辅基和全酶形式的两个活性位点中都观察到了明显的环移位以及氢键网络和范德华相互作用的重新组织，这种结构变化调节是从脱辅基-sGDH的精细X射线结构（PDB：5MIN）中揭示出来的。
