CONFORMATIONAL MODELING OF SUBSTRATE-BINDING TO ENDOCELLULASE E2 FROMTHERMOMONOSPORA-FUSCA

Molecular mechanics calculations have been used to place a cellotetrao se substrate into the active site of the crystallographically determin ed structure of endocellulase E2 from Thermomonospora fusca. In the lo west energy model structure, the second residue of the substrate oligo saccharide is tilted away from the planar ribbon geometry of cellulose as it is in the X-ray structure of the E2(cd)-cellobiose co-crystal, This tilt is the result of the topology of the binding site, and resul ts In several strong carbohydrate-protein hydrogen bonds, The tilting produces a twisting of the glycosidic linkage of the cleavage site bet ween residues two and three. In the predicted enzyme-substrate complex both of the Asp residues believed to function in general acid and bas e roles in the previously proposed model for the mechanism are distant from the bond being cleaved. Molecular dynamics simulations of the co mplex were conducted, and while the putative catalytic Asp residues re mained distant from the cleavage site, the proton of Tyr73 briefly cam e within van der Waals contact of the linkage oxygen.