Fig. 9: Mechanistic view of the catalytic function of HvExoI, a family GH3 enzyme.

a HvExoI uses the retaining catalytic mechanism, where nucleophile Asp285 (magenta) and acid/base catalyst Glu491 (green) play key roles in substrate hydrolysis^1,2,3. b Reaction kinetics indicates the formation of Michaelis complexes (E-S and E~S), and enzyme-product complexes before (E~P1~Glc) and following the first Glc egress (E~P1), and after hydrolysis of P1 (E~Glc)^{1, 2}. S and P1 are trimeric and dimeric molecules, respectively. First-order rate constants are shown. Entrapped Glc product from E~Glc is displaced after a new substrate is attached. The hydrolytic cycle with a (1,3)-linked substrate is repeated until it is hydrolysed to Glc (indicated by vertical and horizontal lines). c Simplified mechanism of Glc displacement with a disaccharide²². After the disaccharide (empty blue and filled grey squares) bound in the −1 and +1 subsites (step 1) is hydrolysed and the reducing end Glc (aglycon) diffuses away from +1 subsite, the non-reducing end Glc (cyan square) remains non-covalently trapped. After a new dimer binds (step 3) and advances to the catalytic site, Glc modifies its binding patterns and egresses (large arrow) via a transient lateral cavity (cylinder in dotted lines) formed near the Trp clamp (step 4)²². Curved arrows indicate dimer entries into the catalytic site. d Glc displacement paths in WT (left panels), and the W434A mutant (right panels) with Glc and the G3OG dimer, calculated through cMD and GPathFinder. Large arrows indicate Glc positions at the initial (step 1) and after the final (step 4 – following Glc departure from −1 subsites) stages of catalysis (this work).