title: Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.
abstract: The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc.
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University, NSM, 1 Universitetsvej, Build. 28, DK-4000 Roskilde, Denmark.
authors.forename: Johan P
authors.initials: JP
authors.lastname: Olsen
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University, NSM, 1 Universitetsvej, Build. 28, DK-4000 Roskilde, Denmark.
authors.forename: Kadri
authors.initials: K
authors.lastname: Alasepp
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University, NSM, 1 Universitetsvej, Build. 28, DK-4000 Roskilde, Denmark.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Kari
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University, NSM, 1 Universitetsvej, Build. 28, DK-4000 Roskilde, Denmark.|Novozymes A/S, Bagsvaerd, Denmark.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: Novozymes A/S, Bagsvaerd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University, NSM, 1 Universitetsvej, Build. 28, DK-4000 Roskilde, Denmark. pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D002482:Cellulose / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D003198:Computer Simulation; D004789:Enzyme Activation; D006868:Hydrolysis; D008956:Models, Chemical; D012995:Solubility
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: biosensor; cellobiohydrolase; cellulase; enzyme kinetics; product inhibition