title: The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.
abstract: Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.
authors.affiliation: From the Institute of Molecular and Cell Biology, University of Tartu, 51010 Tartu, Estonia and.
authors.forename: Silja
authors.initials: S
authors.lastname: Kuusk
authors.affiliation: the Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
authors.affiliation: From the Institute of Molecular and Cell Biology, University of Tartu, 51010 Tartu, Estonia and priit.valjamae@ut.ee.
authors.forename: Priit
authors.initials: P
authors.lastname: Väljamäe
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D049329:Nanostructures; D011134:Polysaccharides / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D012706:Serratia marcescens / Q000201:enzymology
chemical_list: D004187:Disaccharides; D011134:Polysaccharides; D002686:Chitin; C010913:N,N-diacetylchitobiose; D002482:Cellulose; D002688:Chitinases; C094590:chitinase ChiA; D043366:Cellulose 1,4-beta-Cellobiosidase; C109691:microcrystalline cellulose
keywords: Cellulase; Cellulose; Chitin; Chitinase; Enzyme Kinetics; Product Inhibition; Rate-limiting Step