Abstract
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 HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl β-d-glucoside and methyl 6-thio-β-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-β-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI 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 HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.
Highlights
Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories
Several crystals removed from the mother liquor were extracted and fractionated by normal-phase high performance liquid chromatography (HPLC) with evaporative light scattering detection (Fig. 1a; top panel, solid line)
Most approaches towards the descriptions of reactant movements are based on Molecular dynamics (MD) simulations of substrate and product binding and unbinding, including random collisions or diffusion[40]
Summary
Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. Isolated from barley seedlings, was that the glucose (Glc) product released from β-D-glucoside substrates remains entrapped in the enzyme active site until an incoming substrate binds[6,7,8,9], presumably lowering the energy barrier to facilitate Glc displacement. At this stage, the mechanism of Glc displacement and how it is linked to the catalytic cycle of HvExoI remained unanswered. The native GH78 α-L-rhamnosidase with a deep pocketshaped active site holds the entrapped Glc molecule, which was not perfused in crystals[14]
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