title: Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes.
abstract: Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.
authors: Energy Biosciences Institute, University of California, Berkeley, California 94720, United States.
authors: Jerome M
authors: JM
authors: Fox
authors: Seth E
authors: SE
authors: Levine
authors: Douglas S
authors: DS
authors: Clark
authors: Harvey W
authors: HW
authors: Blanch
mesh_terms: D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D009844:Oligosaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D032901:Talaromyces / Q000201:enzymology / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014306:Trioses / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D014306:Trioses; C506455:beta-1,3-1,4-cellotriose; C099423:cellohexaose; D002482:Cellulose; C056373:endoglucanase 2; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase