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Literature summary for 3.2.1.176 extracted from

  • Nakamura, A.; Tsukada, T.; Auer, S.; Furuta, T.; Wada, M.; Koivula, A.; Igarashi, K.; Samejima, M.
    The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose (2013), J. Biol. Chem., 288, 13503-13510.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain DH5alpha Trichoderma reesei
gene cel7A, recombinant expression of wild-type and mutant W40A enzymes under the control of its own promoter in Trichoderma reesei strain ALKO 3413 lacking the genes encoding for endogenous Cel7A and Cel7B Trichoderma reesei

Protein Variants

Protein Variants Comment Organism
W40A mutation of Trp40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Comparison of activities of the wild-type and mutant W40A enzymes (with and without the cellulose-binding domain) for various substrates, overview Trichoderma reesei
W40A the mutation causes a loss of crystalline cellulose-degrading ability. The mutant shows reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel Trichoderma reesei

Inhibitors

Inhibitors Comment Organism Structure
additional information 4-methylumbelliferyl beta-D-lactoside substrate inhibition Trichoderma reesei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetic Trichoderma reesei
0.293
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme catalytic domain Trichoderma reesei
0.318
-
4-methylumbelliferyl beta-D-lactopyranoside mutant enzyme W40A, at pH 5.0 and 27°C Trichoderma reesei
0.318
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme Trichoderma reesei
0.335
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme catalytic domain Trichoderma reesei
0.358
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme Trichoderma reesei
0.358
-
4-methylumbelliferyl beta-D-lactopyranoside wild type enzyme, at pH 5.0 and 27°C Trichoderma reesei

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Trichoderma reesei the enzyme has the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain are both necessary for full activity on crystalline substrates ?
-
?

Organism

Organism UniProt Comment Textmining
Trichoderma reesei
-
-
-
Trichoderma reesei G0RVK1 gene cel7A
-
Trichoderma reesei ALKO 3413
-
-
-

Purification (Commentary)

Purification (Comment) Organism
Bio-Gel P-6 gel filtration, DEAE-Sepharose column chromatography and phenyl Sepharose column chromatography Trichoderma reesei
recombinant -type and mutant W40A enzymes from Trichoderma reesei strain ALKO 3413 culture filtrate by gel filtration, anion exchange, hydrophobic interaction, and affinity chromatography Trichoderma reesei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-methylumbelliferyl beta-D-cellobioside + H2O
-
Trichoderma reesei 4-methylumbelliferone + cellobiose
-
?
4-methylumbelliferyl beta-D-cellobioside + H2O
-
Trichoderma reesei ALKO 3413 4-methylumbelliferone + cellobiose
-
?
4-methylumbelliferyl beta-D-lactopyranoside + H2O
-
Trichoderma reesei 4-methylumbelliferone + lactose
-
?
4-methylumbelliferyl beta-D-lactopyranoside + H2O
-
Trichoderma reesei ALKO 3413 4-methylumbelliferone + lactose
-
?
4-methylumbelliferyl beta-D-lactoside + H2O
-
Trichoderma reesei 4-methylumbelliferol + D-lactose
-
?
crystalline cellulose + H2O the enzyme shows processive hydrolysis by the catalytic domain and a sliding movement of single enzyme molecules on a highly crystalline cellulose surface, the cellulose-binding domain is unnecessary for the sliding movement, real-time monitoring by high-speed atomic force microscope Trichoderma reesei ?
-
?
highly crystalline cellulose + H2O
-
Trichoderma reesei cellobiose
-
?
highly crystalline cellulose + H2O
-
Trichoderma reesei ALKO 3413 cellobiose
-
?
additional information the enzyme has the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain are both necessary for full activity on crystalline substrates Trichoderma reesei ?
-
?
additional information comparison of activities of the wild-type and mutangt W40A enzymes (with and without the cellulose-binding domain) for various substrates, overview Trichoderma reesei ?
-
?
phosphoric acid-swollen cellulose + H2O
-
Trichoderma reesei cellobiose
-
?
phosphoric acid-swollen cellulose + H2O
-
Trichoderma reesei ALKO 3413 cellobiose
-
?

Subunits

Subunits Comment Organism
More three-dimensional structure of the catalytic domain of TrCel7A cellobiohydrolase, overview Trichoderma reesei

Synonyms

Synonyms Comment Organism
Cel7A
-
Trichoderma reesei
cellobiohydrolase
-
Trichoderma reesei

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.33
-
4-methylumbelliferyl beta-D-lactopyranoside mutant enzyme W40A, at pH 5.0 and 27°C Trichoderma reesei
0.33
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme Trichoderma reesei
0.361
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme catalytic domain Trichoderma reesei
0.415
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme catalytic domain Trichoderma reesei
0.49
-
4-methylumbelliferyl beta-D-lactopyranoside wild type enzyme, at pH 5.0 and 27°C Trichoderma reesei
0.492
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme Trichoderma reesei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5 6 assay at Trichoderma reesei

General Information

General Information Comment Organism
evolution the enzyme belongs to the glycoside hydrolase family 7, GH7 Trichoderma reesei
malfunction mutation of Trp40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Inactive mutant enzyme does not slide on a cellulose surface Trichoderma reesei
additional information three-dimensional structure of the catalytic domain of TrCel7A cellobiohydrolase, Trp40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis, molecular dynamics simulation study, overview. The reducing end glucose unit is effectively loaded into the active site ofWTcat, but not into that of W40Acat, when the simulation is started from subsite-7 Trichoderma reesei

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.03
-
4-methylumbelliferyl beta-D-lactopyranoside mutant enzyme W40A, at pH 5.0 and 27°C Trichoderma reesei
1.03
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme Trichoderma reesei
1.23
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant mutant W40A enzyme catalytic domain Trichoderma reesei
1.23
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme catalytic domain Trichoderma reesei
1.37
-
4-methylumbelliferyl beta-D-lactoside pH 5.0, 27°C, recombinant wild-type enzyme Trichoderma reesei
1.37
-
4-methylumbelliferyl beta-D-lactopyranoside wild type enzyme, at pH 5.0 and 27°C Trichoderma reesei