昼夜节律作为生命体适应地球自转的重要调控系统，其核心的转录-翻译负反馈环路（TTFL）机制在真核生物中展现出惊人的演化保守性。本研究通过多物种比较分析揭示了P-loop ATP水解酶RUVBL2在真核生物生物钟系统中的核心地位，并首次证实慢速ATP酶活性这一原核生物钟特征在真核生物中的功能性延续，为理解生物钟系统的演化起源提供了新的理论框架。

传统观点认为，真核生物通过转录-翻译负反馈环路实现节律振荡，该分子机制在真菌、植物和动物中均表现出保守性。然而，分子演化分析显示不同物种核心生物钟蛋白质序列缺乏同源保守性，这一矛盾暗示着尚未被揭示的深层调控机制。本研究采用跨尺度研究方法，发现哺乳动物生物钟超复合体关键的组分RUVBL2蛋白，其独特的慢速ATP酶活性（每天约水解13个ATP）与蓝藻KaiC ATP酶时钟的计时特性高度相似。通过CRISPR-Cas9介导的基因敲除、shRNA敲低等遗传学操作，结合专一性小分子药物处理（如RUVBL1/2 ATP酶抑制剂CB-6644）以及经腺相关病毒（AAV）递送至小鼠视交叉上核（SCN）的体内靶向调控，我们证实RUVBL2催化活性的改变可精确调控昼夜节律周期。这种基于酶活性速率的计时模式突破了传统TTFL模型的解释范畴，揭示出ATP水解动力学参数与昼夜周期长度之间存在的定量关系。

跨物种研究表明，专一性小分子抑制剂CB-6644均可以有效抑制真核生物RUVBL2同源蛋白，且产生可预测的长周期节律表型。这一发现不仅确立了RUVBL2作为真核生物钟的共有核心元件，更揭示了原核与真核生物钟在分子机制层面的深层次功能性趋同演化。特别值得关注的是，ATP水解速率的数量级差异（典型ATP酶：~10²-10³/s vs RUVBL2：~10^-2/s）提示生物钟系统可能通过“分子节流阀”机制实现精确计时。

最后，我们结合P-loop ATP酶在原子层面的水解特性，系统研究了相对于绝大多数典型ATP酶的快速水解活性，慢速P-loop ATP酶KaiC和RUVBL2如何在原子层面调控自身的慢速ATP水解的特性，并经腺相关病毒递送至小鼠视交叉上进行了体内靶向调控的验证。这一理论模型的建立，为理解ATP酶的功能演化提供了理论基础。

本研究的突破性发现体现在三个层面：首先，阐明哺乳动物核心生物钟蛋白RUVBL2通过其特有的慢速催化活性直接参与节律周期调控；其次，证实ATP酶计时模块在真核生物钟中的功能性保守；最后，建立慢速P-loop ATP酶的原子层面的水解特性。这些发现不仅为生物钟分子机制的演化研究提供了关键实验证据，更为节律相关疾病的靶向治疗开辟了新途径。后续研究将聚焦于解析RUVBL2酶活性调控网络及其与经典TTFL系统的耦合机制。

Circadian rhythms, as a crucial regulatory system for organisms to adapt to Earth's rotation, exhibit remarkable evolutionary conservation in their core transcriptional-translational negative feedback loop (TTFL) mechanism across eukaryotes. This study reveals through multispecies comparative analysis the central role of the P-loop ATP hydrolase RUVBL2 in eukaryotic circadian systems, and for the first time demonstrates the functional continuity of slow ATPase activity—a hallmark of prokaryotic circadian clocks—in eukaryotes, establishing a novel theoretical framework for understanding the evolutionary origins of circadian systems. While traditional views posit that eukaryotic circadian oscillations are achieved through TTFL mechanisms showing structural conservation across fungi, plants, and animals, molecular evolutionary analyses reveal a paradoxical lack of sequence homology in core clock proteins. This discrepancy suggests undiscovered deep regulatory mechanisms. Our cross-scale investigation identifies the mammalian circadian super complex component RUVBL2, whose unique slow ATPase activity (hydrolyzing ~13 ATP molecules daily) mirrors the timekeeping characteristics of cyanobacterial KaiC ATPase. Through CRISPR-Cas9-mediated knockout, shRNA knockdown, and the specific pharmacological modulation (e.g., RUVBL1/2 ATPase inhibitor CB-6644), and AAV-mediated delivery to mouse suprachiasmatic nucleus (SCN), we demonstrate that alterations in RUVBL2 catalytic activity precisely regulate circadian period, revealing a quantitative relationship between ATP hydrolysis kinetics and period length that transcends traditional TTFL paradigms. Cross-species studies show that specific inhibitor CB-6644 effectively targets eukaryotic RUVBL2 homologs, producing predictable long-period phenotypes. These findings not only establish RUVBL2 as a conserved core element in eukaryotic clocks but also reveal deep functional convergence between prokaryotic and eukaryotic timing systems. Notably, the orders-of-magnitude difference in ATP hydrolysis rates (canonical ATPases: ~102-103/s vs RUVBL2: ~10-2/s) suggests a "molecular throttle" mechanism for precise timekeeping. Furthermore, we elucidated the hydrolysis characteristics of P-loop ATPases at the atomic level. Through systematic investigation, we deciphered how slow P-loop ATPases KaiC and RUVBL2 regulate their intrinsically slow hydrolytic activity at the atomic scale, compared to the rapid hydrolysis exhibited by the majority of canonical ATPases. This mechanistic insight was further validated through in vivo targeted modulation experiments via adeno-associated virus (AAV)-mediated delivery to the mouse suprachiasmatic nucleus (SCN). The establishment of this theoretical model provides a foundational framework for understanding the functional evolution of ATPases. The groundbreaking discoveries of this study are manifested in three key aspects: First, we elucidate how the mammalian core clock protein RUVBL2 directly regulates circadian period through its unique slow catalytic activity. Second, we demonstrate the functional conservation of ATPase timing modules in eukaryotic circadian systems. Finally, we establish the atomic-level hydrolysis characteristics of slow P-loop ATPases. These findings not only provide crucial experimental evidence for understanding the molecular evolution of circadian mechanisms but also open new avenues for targeted therapies in circadian rhythm-related disorders. Future research will focus on deciphering the RUVBL2 enzymatic regulatory network and its coupling with the classical TTFL system.