Observation of Proton-Driven Activity Fluctuations Reveals the Catalytic Rhythm of Single-Enzymes

Abstract Single enzymes exhibit fluctuations in catalytic activity over time, determining the functionality of enzyme-regulated biological processes. These fluctuations are perceived as random and cannot be explained by classic Markovian dynamics. Here, we observed rhythmic catalytic behavior of single enzymes at microsecond level by viscosity-confined single-entity electrochemistry of collision (VC-SEEC). Enzymes transition between resting and working states with “clock-like” regularity, revealing catalytic rhythms (~ 80–150 Hz) of glucose oxidase and acetylcholinesterase. Resting time typically exceeds working time by over 4-fold, which dominates the rhythm. Remarkably, kinetic and thermodynamic analyses unveil tunable catalytic rhythms, enabling more than 10-fold improvement in performance by accelerating the rhythm 3.1 times. Combined experimental and simulation results further reveal that proton diffusion generated during catalysis is an important contributor to the rhythmic behavior. This insight would transform our understanding of enzyme catalysis mechanisms, offering valuable insights for precision medicine and directed enzyme evolution.