The titration of the active centers of cellobiohydrolase from Trichoderma reesei

Rabinowitch, M. L.; Klyosov, Anatole A.; Melnick, M S

doi:10.1016/0003-2697(86)90283-6

Cited by 8 publications

(6 citation statements)

References 4 publications

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“…Interaction (inhibition) constants of ligands were determined conventionally (1/v vs [I] plots) or by active site titrations as described in [17], using the same lactoside as indicator substrate. Avicel suspensions (30 mg" m1-1 in 50 mM sodium acetate/acetic acid, pH 5.0) were used to study the activity (50°C) and adsorption (20°C) of CBH I against and onto this substrate as described [9].…”

Section: Enzymes and Enzymic Assaysmentioning

confidence: 99%

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Tomme

Claeyssens

1989

FEBS Letters

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Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-Y-sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pKa of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125-135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.

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Section: Enzymes and Enzymic Assaysmentioning

confidence: 99%

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Tomme

Claeyssens

1989

FEBS Letters

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“…Calculation approach is addressed in detail in Section 8 of Supporting Information. The obtained results are compared with the literature data in Table .…”

Section: Resultsmentioning

confidence: 96%

“…refs. ). What are the k cat / K m values that are insensitive to other‐than‐Michaelis complex intermediates and allow evaluation of the contribution of separate subsites and comparison with the other cellulases?…”

Section: Resultsmentioning

confidence: 97%

“…However, both strength and the type of cellobiose inhibition of HjCel7A substantially depend on the nature of substrate . Although this phenomenon has been established for more than 30 years, it was readdressed in detail in a series of publications since 2004 …”

Section: Resultsmentioning

confidence: 99%

“…High‐affinity nonproductive complexes of CBH I with both soluble (MUG 2 , cellooligosaccharides, Na‐CMC), and insoluble substrates were also kinetically documented in the catalytic pathway of CBH I. Complexes with cellulose could not be decomposed by an excess of soluble ligands: CMC, cellobiose, or MUL . Structural data revealed a series of complexes of cellobihexaose with downstream (+1, +2, product‐binding), upstream (−7 to −2, substrate‐binding) subsites or ternary nonproductive complexes with both downstream and upstream subsites of inactive HjCel7A mutants.…”

Section: Introductionmentioning

confidence: 96%

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Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

et al. 2018

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Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes nine subsites for β-d-glucose units, seven of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k /K for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG ). Interaction of substrates with the subsites -6 and -5 proximal to the nonconserved Gln101 residue in HjCel7A decreases K by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K ≈2-9 nM via CBM and CD that captures six terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding.

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Application of cellulose‐based self‐assembled tri‐enzyme system in a pseudo‐reagent‐less biosensor for biogenic catecholamine detection

Rabinovich

Vasil'chenko

Karapetyan

et al. 2007

Biotechnology Journal

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Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.

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The titration of the active centers of cellobiohydrolase from Trichoderma reesei

Cited by 8 publications

References 4 publications

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

Application of cellulose‐based self‐assembled tri‐enzyme system in a pseudo‐reagent‐less biosensor for biogenic catecholamine detection

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