title: Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.
abstract: Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.
authors.affiliation: From the Institutes of Molecular and Cell Biology and.
authors.forename: Mihhail
authors.initials: M
authors.lastname: Kurašin
authors.affiliation: From the Institutes of Molecular and Cell Biology and.
authors.forename: Silja
authors.initials: S
authors.lastname: Kuusk
authors.affiliation: Physics, University of Tartu, 51010 Tartu, Estonia and.
authors.forename: Piret
authors.initials: P
authors.lastname: Kuusk
authors.affiliation: the Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås 1432, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
authors.affiliation: From the Institutes of Molecular and Cell Biology and priit.valjamae@ut.ee.
authors.forename: Priit
authors.initials: P
authors.lastname: Väljamäe
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D020125:Mutation, Missense; D011485:Protein Binding; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002686:Chitin; D002688:Chitinases; C000601656:chitinase A, Serratia marcescens
keywords: cellobiohydrolase; cellulose; chitin; chitinase; off-rate; processivity; product binding