Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into α-d-glucose 1-phosphate (G1P) and d-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites -1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy ⁴C₁ conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from ¹S₃ as the pre-transition state conformer (Michaelis complex) via E₃ as the transition state candidate to ⁴C₁ as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The ¹S₃ pre-transition state conformer is highly stable compared with other conformers, and a conformational change from ⁴C₁ to ^1,4B was observed.