We investigated the catalytic mechanism of α-1,4-glucan lyases using a full QM DFT approach based on the M06-2X functional. The reaction profile of the whole catalytic process can be divided into three phases: glycosylation, deglycosylation-elimination and tautomerization. Glycosylation is a highly asynchronous SN 1-like process with an energy barrier of 10.2 kcal mol-1 . A proton moves from the Asp665 residue to the glycosidic oxygen. Asp553 acts as a nucleophile and attacks the anomeric carbon causing the cleavage of the glycosidic bond. Deglycosilation-elimination is the rate-determining step of the entire process with an overall barrier of 18.3 kcal mol-1 . The final step (restoring the catalyst and tautomerization) occurs rather easily, since the Asp553 carboxylate group "assists" the proton transfer in the tautomerization process. Our computations clearly indicate that tautomerization must occur inside the enzyme before leaving the active site rather than in the aqueous solution. Outside of the protein environment the enol-AF→keto-AF process "assisted" by a water molecule has a barrier of 35.8 kcal mol-1 .
Keywords: DFT calculations; glycoside hydrolases; glycosylation; reaction mechanism; tautomerization.
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