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Literature summary for 3.2.1.4 extracted from

  • Zhou, W.; Irwin, D.C.; Escovar-Kousen, J.; Wilson, D.B.
    Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes (2004), Biochemistry, 43, 9655-9663.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
12 mutant genes with changes in fice conserved residues of Cel9A-68 are constructed, cloned and expressed in Escherichia coli Thermobifida fusca

Protein Variants

Protein Variants Comment Organism
D55A activity with carboxymethyl cellulose is 0.2% of wild-type activity, activity with phosphoric acid-swollen cellulose is 1.2% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 1.6% of wild-type activity Thermobifida fusca
D55A/D58A activity with carboxymethyl cellulose is less than 0.1% of wild-type activity, activity with phosphoric acid-swollen cellulose is 0.13% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 0.5% of wild-type activity Thermobifida fusca
D55N activity with carboxymethyl cellulose is 0.3% of wild-type activity, activity with phosphoric acid-swollen cellulose is 1.6% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 2.2% of wild-type activity Thermobifida fusca
D58A activity with carboxymethyl cellulose is 0.4% of wild-type activity, activity with phosphoric acid-swollen cellulose is 1.8% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 3.5% of wild-type activity, mutant enzyme loses about 90% of the initial activity after 15 h at 65°C, compared to 10% loss of wild-type activity Thermobifida fusca
D58N activity with carboxymethyl cellulose is 0.45% of wild-type activity, activity with phosphoric acid-swollen cellulose is 2% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 2.7% of wild-type activity, mutant enzyme loses about 20% of the initial activity after 15 h at 65°C, compared to 10% loss of wild-type activity Thermobifida fusca
DELTAT245-L251/R252K activity with carboxymethyl cellulose, acid-swollen cellulose or bacterial microcrystalline cellulose from Acetobacter xylinum is nearly identical to wild-type activity Thermobifida fusca
E424A activity with carboxymethyl cellulose is 0.13% of wild-type activity, activity with phosphoric acid-swollen cellulose is 0.2% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 0.43% of wild-type activity, kcat/Km for 2,4-dinitrophenyl beta-D-cellobioside is 8.5fold higher than the wild-type value Thermobifida fusca
E424G activity with carboxymethyl cellulose is 0.3% of wild-type activity, activity with phosphoric acid-swollen cellulose is 1.1% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 2.5% of wild-type activity, kcat/Km for 2,4-dinitrophenyl beta-D-cellobioside is 123.8fold higher than the wild-type value Thermobifida fusca
E424Q activity with carboxymethyl cellulose is less than 0.1% of wild-type activity, activity with phosphoric acid-swollen cellulose is 0.15% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 1.1% of wild-type activity Thermobifida fusca
additional information the FnIII-like domain is deleted from Cel19A-90, reducing activity with bacterial microcrystalline cellulose to 43% of the wild-type Thermobifida fusca
Y206F activity with carboxymethyl cellulose is 7% of wild-type activity, activity with phosphoric acid-swollen cellulose is 4.1% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 8.3% of wild-type activity, mutant enzyme loses about 20% of the initial activity after 15 h at 65°C, compared to 10% loss of wild-type activity Thermobifida fusca
Y206S activity with carboxymethyl cellulose is 0.5% of wild-type activity, activity with phosphoric acid-swollen cellulose is 1.3% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 1.8% of wild-type activity Thermobifida fusca
Y318A activity with carboxymethyl cellulose is 5fold higher than wild-type activity, activity with phosphoric acid-swollen cellulose is 28% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 14.2% of wild-type activity, mutant enzyme loses about 30% of the initial activity after 15 h at 65°C, compared to 10% loss of wild-type activity Thermobifida fusca
Y318F activity with carboxymethyl cellulose is 6.7fold higher than wild-type activity, activity with phosphoric acid-swollen cellulose is 75% of wild-type activity, activity with bacterial microcrystalline cellulose from Acetobacter xylinum is 16.6% of wild-type activity Thermobifida fusca

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.197
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, mutant enzyme E424G Thermobifida fusca
0.29
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, mutant enzyme E424A Thermobifida fusca
0.697
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, wild-type enzyme Cel9A-68 Thermobifida fusca

Organism

Organism UniProt Comment Textmining
Thermobifida fusca P26221 endoglucanase E-4 precursor; Cel9A
-
Thermobifida fusca Cel9A P26221 endoglucanase E-4 precursor; Cel9A
-

Purification (Commentary)

Purification (Comment) Organism
-
Thermobifida fusca

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,4-dinitrophenyl beta-D-cellobioside + H2O
-
Thermobifida fusca ?
-
?
2,4-dinitrophenyl beta-D-cellobioside + H2O
-
Thermobifida fusca Cel9A ?
-
?
bacterial microcrystalline cellulose + H2O
-
Thermobifida fusca ?
-
?
bacterial microcrystalline cellulose + H2O
-
Thermobifida fusca Cel9A ?
-
?
carboxymethyl cellulose + H2O
-
Thermobifida fusca ?
-
?
carboxymethyl cellulose + H2O
-
Thermobifida fusca Cel9A ?
-
?
phosphoric acid-swollen cellulose + H2O
-
Thermobifida fusca ?
-
?
phosphoric acid-swollen cellulose + H2O
-
Thermobifida fusca Cel9A ?
-
?

Synonyms

Synonyms Comment Organism
Cel9A-68 Cel9A is secreted as a 90400 Da protein. Limited proteolysis of Cel9A produces Cel9A-68 Thermobifida fusca
Cel9A-90 Cel9A is secreted as a 90400 Da protein. Limited proteolysis of Cel9A produces Cel9A-68 Thermobifida fusca

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
15 h, wild-type enzyme and mutant enzymes Y206F, D58A and Y318A are stable, mutant enzyme D58N loses about 20% of its inital activity Thermobifida fusca
60
-
15 h, wild-type enzyme is stable, mutant enzyme D58A loses about 20% of initial activity, mutant enzymes Y206F, D58N, D58N and Y318A lose about 10% of the inital activity Thermobifida fusca
65
-
15 h, wild-type enzyme loses about 10% of initial activity, mutant enzyme D58N and Y206F loses about 20% of maximal activity, mutant enzyme D58A loses about 90% of initial activity, mutant enzyme Y318A loses about 30% of initial activity Thermobifida fusca
70
-
wild-type enzyme loses about 50% of initial activity, mutant enzyme D58A loses about 20% of initial activity, mutant enymes D58A, Y206F and Y318A nearly completely lose activity Thermobifida fusca

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, wild-type enzyme Cel9A-68 Thermobifida fusca
0.07
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, mutant enzyme E424A Thermobifida fusca
0.7
-
2,4-dinitrophenyl beta-D-cellobioside 50°C, mutant enzyme E424G Thermobifida fusca