Molecular Details from Computational Reaction Dynamics for the Cellobiohydrolase I Glycosylation Reaction

Abstract

Glycosylation of cellobiose hydrolase I (CBHI), is a key step in the processing and degradation of cellulose. Here the pathways and barriers of the reaction are explored using the free energy from adaptive reaction coordinate forces (FEARCF) reaction dynamics method coupled with SCC-DFTB/MM. In many respects CBHI follows the expected general GH7 family mechanism that involves the Glu-X-Asp-X-X-Glu motif. However, critical electronic and conformational details, previously not known, were discovered through our computations. The central feature that ensures the success of the glycosylation reaction are the Glu212 nucleophile's hydrogen bond to the hydroxyl on C2, of the glucose in the -1 position of the cellulosic strand. This Glu212 function restricts the C2 hydroxyl in such a way as to favor the formation of the (4)E ring pucker of the -1 position glucose. A frontier molecular orbital analysis of the structures along the reaction surface proves the existence of an oxocarbenium ion, which has both transition state and intermediate character. The transition state structure is able to descend down the glycosylation pathway through the critical combination of Asp214 (HOMO), ring oxygen (LUMO), and Glu212 (HOMO), anomeric carbon (LUMO) interactions. Using the fully converged FEARCF SCC-DFTB/MM reaction surface, we find a barrier of 17.48 kcal/mol separating bound cellulose chain from the glycosylated CBHI. Taking recrossing into account gives k(cat) = 0.415 s(-1) for cellobiohydrolase glycosylation.

Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose

“…Catalysis-The free energy barrier for catalysis was about 69 kJ/mol at room temperature, and this is in good accordance with theoretical studies, which have found ⌬G cat values of 65 and 73 kJ/mol (12,14). The entropic contribution was dominating (ϪT⌬S cat ϭ 40 kJ/mol), although ⌬H cat was only moderately smaller (29 kJ/mol).…”

“…This theoretical work has provided important information on the structure and processive movement of the cellulose strand in the 5-nm-long binding tunnel, which houses the active site, and suggested (12,14) that formation of the intermediate (so-called glycosylation) is the rate-limiting step in the catalytic reaction sequence. One study computed a catalytic rate constant around 11 s Ϫ1 for glycosylation (14), and this is in line with experimental attempts to single out k cat for Cel7A acting on insoluble cellulose (15)(16)(17)(18).…”

FEARCF a multidimensional free energy method for investigating conformational landscapes and chemical reaction mechanisms

“…Although the three dimensional free energy volume of the glucose ring pucker is very complicated we have shown that we are able to traverse the entire conformational space of carbohydrate ring pucker. We show in subsequent publications that this is critical to investigations of glycosidase reactions [61]. NAIDOO Kevin J. holds a PhD from the University of Michigan (USA) and following this he was a Postdoctoral Fellow (1994)(1995) at Cornell University (USA).…”

Cellulase biocatalysis: key influencing factors and mode of action

“…A third acidic amino acid residue called aspartic acid (D214) has been reported to assist the nucleophilic attack. The mutations of these two active site amino acids have shown the reduction of rate of hydrolysis by 85-370 fold (Barnett et al 2011;Li et al 2010;Yan et al 2011 (Zhang et al 2013). TrCel6A, glycoside hydrolases (GH6 and GH7) TrCel6A is a processive cellulase and emphasized the requirement for accurate GH6 processivity assays (Payne et al 2015) GH6 demonstrated higher hydrolysis activity towards homoglycosidic linkage of cellulose polymers, but relatively poor hydrolysis of the heteroglycosidic linkages Turnover of GH6 of these molecules was remained slow when compared to GH7 activity Fig.…”