Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExol isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl beta-D-glucoside and methyl 6-thio-beta-gentiobioside substrate analogues perfused in crystalline HvExol bind across the catalytic site after they displace glucose, while methyl 2-thio-beta-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExol reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExol losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.