α-1,4-Glucan lyase(GLase EC 4.2.2.13),an interesting member of glycoside hydrolase family 31(GH31),specifically cleaves α-1,4-glucosidic bonds in glycogen,starch and malto-oligosaccharides to produce 1,5-anhydro-D-fructose(AF)from the non-reducing end.Previous studies have proved that GLase is a retaining glycoside lyase[1],whose catalytic cycle involves two distinct steps(glycosylation and deglycosylation),with a covalent glycosyl-enzyme intermediate formed.Subsequently,the glycosidic bond of this intermediate breaks via a β-elimination reaction[2].However,the detailed mechanism remains to be unclear.Here,we used combined quantum-mechanical/molecular-mechanical(QM/MM)approach to investigate the details of the catalytic reaction.Our calculations indicate that the whole catalytic cycle contains five elementary steps.The proton abstraction occurring at the C2 position in deglycosylation step is considered to be rate-determining with an energy barrier of 19.60 kcal/mol.The residue Asp553 was found to directly participate in the proton abstraction.