Recent experiments have revealed that the diffusivity of exothermic and fast enzymes is enhanced when they are catalytically active, and different physical mechanisms have been explored and quantified to account for this observation. We perform measurements on the endothermic and relatively slow enzyme aldolase, which also shows substrate-induced enhanced diffusion. We propose a new physical paradigm, which reveals that the diffusion coefficient of a model enzyme hydrodynamically coupled to its environment increases significantly when undergoing changes in conformational fluctuations in a substrate-dependent manner, and is independent of the overall turnover rate of the underlying enzymatic reaction. Our results show that substrate-induced enhanced diffusion of enzyme molecules can be explained within an equilibrium picture, and that the exothermicity of the catalyzed reaction is not a necessary condition for the observation of this phenomenon.In a quest for understanding nonequilibrium processes encountered in biology and chemistry, the study of active matter, namely systems constituted of agents able to consume and convert energy extracted from their environment, has been a major focus of the contemporary physical sciences [1,2]. Recent progress led to the design, fabrication and characterization of synthetic micro-and nano-machines relying on different propulsion mechanisms, and able to reproduce functions inspired from molecular biology, such as cargo transport or chemical sensing [3,4]. Such autonomous objects could have major technological applications, provided that they are small enough and fully biocompatible. In this context, and going down in scale, enzyme molecules have received a lot of attention, as models of biological nanoscale transducers able to convert chemical energy into mechanical work. Biomolecules typically perform cyclic turnovers in which they bind to substrates and catalytically convert them to products while undergoing conformational changes [5][6][7][8]. Recently, in vitro studies of enzymes using fluorescence correlation spectroscopy (FCS) have revealed that their diffusion coefficient is enhanced in a substrate-dependent manner [9][10][11][12], and that the diffusion enhancement ∆D at substrate saturation was typically of the order of the bare diffusion coefficient of the enzyme D 0 measured in the absence of substrate molecules. This observation holds for a wide range of enzymes, which typically catalyze fast and exothermic chemical reactions, with reaction enthalpies that can reach 40k B T per molecule and catalytic rates up to ∼ 10 4 s −1 for the particular case of catalase [12].This intriguing phenomenon, that could have major implications in the spatial organisation of biological processes [13], was subsequently investigated from a theoretical point of view. It was first suggested that the enhancement of the * P.I. and X.Z. contributed equally to this work. † Present address: Indian Institute of Technology Gandhinagar, Palaj Campus, Gandhinagar, Gujarat 382 355, India. ‡ Corre...