Catalytic mechanism of a plant hydrolytic enzyme explained through its 50 crystal structures and counting – What have we learnt?

Substrates and products bind and unbind in enzymes at prohibitive rates, which preclude investigations of reactant trajectories during catalytic cycles. To offset this lack of knowledge, we provide data based on 50 crystal structures of a plant exo-hydrolase family 3 enzyme and combine it with kinetics, structural modelling, and evolutionary-based considerations. We demonstrate that in this exo-hydrolase, a non-covalently trapped glucose product induces the formation of a transient lateral cavity, facilitating glucose egress and initiating a subsequent catalytic cycle. This path enables substrate-product-assisted processivity, whilst the enzyme maintains contact with the substrate. Evaluations of nanoscale reactant movements of this enzyme, mutated in an aromatic clamp facing the active site, define reactant trajectories critical to the conformational behaviour of isomeric β-d-glucosides. Additionally, computational models define the role of an essential, non-nucleophilic glutamate 220, which does not participate directly in catalysis but maintains a water molecule network. These models describe the dynamics of water molecule flux and correlate with high catalytic efficiency in the wild-type but not in the Glu220Ala mutant. Evolutionary considerations of the glycoside hydrolase family 3 reveal the evolvability of structural elements and provide a blueprint for the mechanism and dynamics of catalysis in hydrolytic enzymes - a knowledge applicable to life sciences and biotechnologies to safeguard a sustainable bioeconomy.