title: The slowdown of the endoglucanase Trichoderma reesei Cel5A-catalyzed cellulose hydrolysis is related to its initial activity.
abstract: One important feature of hydrolysis of cellulose by cellulases is that the reaction slows down quickly after it starts. In this work, we investigate the slowdown mechanism at the early stage of the reaction using endoglucanase Tr. Cel5A-catalyzed phosphate acid-swollen cellulose (PASC) hydrolysis as a model system. Specifically, we focus on the effect of enzyme adsorption on the reaction slowdown. Nineteen single mutations are introduced (with the assistance of molecular dynamics simulations) to perturb the enzyme PASC interaction, yielding the adsorption partitioning coefficient Kr that ranged from 0.12 to 0.39 L/g, compared to that of the wild type (0.26 L/g). Several residues, including T18, K26, Y26, H229, and T300, are demonstrated to be important for adsorption of the enzyme to PASC. The kinetic measurements show that the slowdown of the hydrolysis is not correlated with the adsorption quantified by the partitioning coefficient Kr but is anticorrelated with the initial activity. This result suggests that the mutants with higher activity are more prone to being trapped or deplete the most reactive substrate faster and the adsorption plays no apparent role in the reaction slowdown. The initial activity of Cel5A against PASC is correlated with the enzyme specific activity against a soluble substrate p-nitrophenyl cellobioside.
authors.affiliation: Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences , Qingdao 266061, China.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Shu
authors.forename: Yefei
authors.initials: Y
authors.lastname: Wang
authors.forename: Liaoyuan
authors.initials: L
authors.lastname: An
authors.forename: Lishan
authors.initials: L
authors.lastname: Yao
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002482:Cellulose; C407024:endoglucanase Cel5A; D002480:Cellulase