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dc.contributor.authorJitrayut Jitonnomen_US
dc.contributor.authorVannajan S. Leeen_US
dc.contributor.authorPiyarat Nimmanpipugen_US
dc.contributor.authorHeather A. Rowlandsen_US
dc.contributor.authorAdrian J. Mulhollanden_US
dc.description.abstractFamily 18 chitinases catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin. The mechanism has been proposed to involve the formation of an oxazolinium ion intermediate via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile (instead of an enzyme residue). Here, we have modeled the first step of the chitin hydrolysis catalyzed by Serratia marcescens chitinase B for the first time using a combined quantum mechanics/molecular mechanics approach. The calculated reaction barriers based on multiple snapshots are 15.8-19.8 kcal mol-1[B3LYP/6-31+G(d)//AM1-CHARMM22], in good agreement with the activation free energy of 16.1 kcal mol-1derived from experiment. The enzyme significantly stabilizes the oxazolinium intermediate. Two stable conformations (4C1-chair and B3,O-boat) of the oxazolinium ion intermediate in subsite -1 were unexpectedly observed. The transition state structure has significant oxacarbenium ion-like character.The glycosyl residue in subsite-1was found to follow a complex conformational pathway during the reaction (1,4B → [4H5/4E]‡→4C1↔ B3,O), indicating complex conformational behavior in glycoside hydrolases that utilize a substrate-assisted catalytic mechanism. TheD142N mutant is found to follow the same wild-type-like mechanism: The calculated barriers for reaction in this mutant (16.0-21.1 kcal mol-1) are higher than in the wild type, in agreement with the experiment. Asp142 is found to be important in transition state and intermediate stabilization. © 2011 American Chemical Society.en_US
dc.subjectBiochemistry, Genetics and Molecular Biologyen_US
dc.titleQuantum mechanics/molecular mechanics modeling of substrate-assisted catalysis in family 18 chitinases: Conformational changes and the role of Asp142 in catalysis in ChiBen_US
article.volume50en_US Mai Universityen_US of Bristolen_US
Appears in Collections:CMUL: Journal Articles

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