Dehydrogenases (DHs) are widely explored bioelectrocatalysts in the development of enzymatic bioelectronics like biosensors and biofuel cells. However, the relatively low intrinsic reaction rates of DHs which mostly depend on diffusional coenzymes (e.g., NAD⁺) have limited their bioelectrocatalytic performance in applications such as biosensors with a high sensitivity. In this study, we find that rare-earth elements (REEs) can enhance the activity of NAD⁺-dependent glutamate dehydrogenase (GDH) toward highly sensitive electrochemical biosensing of glutamate in vivo. Electrochemical studies show that the sensitivity of the GDH-based glutamate biosensor is remarkably enhanced in the presence of REE cations (i.e., Yb³⁺, La³⁺ or Eu³⁺) in solution, of which Yb³⁺ yields the highest sensitivity increase (ca. 95%). With the potential effect of REE cations on NAD⁺ electrochemistry being ruled out, homogeneous kinetic assays by steady-state and stopped-flow spectroscopy reveal a two-fold enhancement in the intrinsic reaction rate of GDH by introducing Yb³⁺, mainly through accelerating the rate-determining NADH releasing step during the catalytic cycle. In-depth structural investigations using small angle X-ray scattering and infrared spectroscopy indicate that Yb³⁺ induces the backbone compaction of GDH and subtle β-sheet transitions in the active site, which may reduce the energetic barrier to NADH dissociation from the binding pocket as further suggested by molecular dynamics simulation. This study not only unmasks the mechanism of REE-promoted GDH kinetics but also paves a new way to highly sensitive biosensing of glutamate in vivo.

中文翻译：

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稀土元素在增强 NAD+ 依赖性谷氨酸脱氢酶生物传感的生物电催化作用中的作用

脱氢酶 (DHs) 是生物传感器和生物燃料电池等酶促生物电子学开发中被广泛探索的生物电催化剂。然而，DHs 相对较低的内在反应速率主要依赖于扩散辅酶（例如NAD ⁺），这限制了它们在高灵敏度生物传感器等应用中的生物电催化性能。在这项研究中，我们发现稀土元素 (REE) 可以增强 NAD ⁺依赖性谷氨酸脱氢酶 (GDH) 对体内谷氨酸的高灵敏度电化学生物传感的活性。电化学研究表明，基于 GDH 的谷氨酸生物传感器的灵敏度在 REE 阳离子（即、Yb ³⁺、La ³⁺或Eu ³⁺ )，其中Yb ³⁺产生最高的灵敏度增加（约95%）。由于排除了REE 阳离子对 NAD ⁺电化学的潜在影响，稳态和停流光谱的均相动力学分析表明，通过引入 Yb ³⁺，GDH 的固有反应速率提高了两倍，主要是通过加速催化循环期间决定速率的 NADH 释放步骤。使用小角 X 射线散射和红外光谱的深入结构研究表明 Yb ³⁺诱导 GDH 的骨架压实和活性位点中微妙的 β-折叠转变，这可能会降低 NADH 从结合口袋解离的能量障碍，正如分子动力学模拟所进一步表明的那样。这项研究不仅揭示了 REE 促进 GDH 动力学的机制，而且还为体内谷氨酸的高灵敏度生物传感铺平了道路。