Proton Tunneling Accelerates ATP Hydrolysis in Eg5 Kinesin

It is well established that, for ATP hydrolysis, a proton from the water nucleophile is abstracted to create a hydroxide capable of attacking the substrate. Evidence for the physical basis for the catalytic power of this key reaction in biological systems has been limited until recently. X‐ray crystal structures capturing this initial step in biological ATP hydrolysis have revealed a two‐water cluster, with uncommon properties, in the active site of kinesin proteins. Herein, we tested the nature of the active‐site water cluster using solvent kinetic isotope experiments. The magnitude of the isotope effect, Arrhenius pre‐exponential factor ratios, and activation energy differences with human Kinesin‐5 proteins were determined. Our data show that the proton transfer between solvent molecules is not classical in description, but rather has a quantum mechanical tunneling component. Experiments with Drosophila Kinesin‐5 showed similar findings; moreover, X‐ray structures of the Drosophila Kinesin‐5 also captured two waters in the active site. Taken together, this is the first detection of enzymatic proton tunneling in an ATPase. More broadly, this study reveals that use of quantum biochemistry is not solely a characteristic of metalloenzymes and is more widespread than currently established. This work was supported by NIH GM097350 (SK).