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Literature summary for 2.4.1.20 extracted from

  • Hamura, K.; Saburi, W.; Matsui, H.; Mori, H.
    Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis (2013), Carbohydr. Res., 379, 21-25.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Ruminococcus albus

Protein Variants

Protein Variants Comment Organism
C485A the mutant shows 43% of wild type activity with cellobiose. The mutant shows higher preference for D-glucosamine than the wild type enzyme Ruminococcus albus
E654A inactive Ruminococcus albus
E654D inactive Ruminococcus albus
Y648A inactive Ruminococcus albus
Y648F The mutant shows 23% of wild type activity with cellobiose. Apparent kcat/Km values of the mutant for D-mannose and 2-deoxy-D-glucose are 8.2 and 4.0fold higher than those of the wild type enzyme, respectively Ruminococcus albus
Y648V the mutant shows 0.888% of wild type activity with cellobiose and has synthetic activity toward N-acetyl-D-glucosamine Ruminococcus albus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.46
-
alpha-D-glucose 1-phosphate wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
1.68
-
cellobiose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
11.3
-
alpha-D-glucose 1-phosphate mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
11.8
-
D-mannose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
13.3
-
D-glucosamine wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
15.7
-
alpha-D-glucose 1-phosphate mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
16.6
-
cellobiose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
17.4
-
N-acetyl-D-glucosamine mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
23.5
-
D-mannose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
30.7
-
alpha-D-glucose 1-phosphate mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
31.4
-
cellobiose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
58.1
-
D-glucosamine mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
60.2
-
2-deoxy-D-glucose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
62.6
-
D-mannose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
83.1
-
2-deoxy-D-glucose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
164
-
2-deoxy-D-glucose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
200
-
D-mannose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
alpha-D-glucose 1-phosphate + D-glucose Ruminococcus albus
-
cellobiose + phosphate
-
r
cellobiose + phosphate Ruminococcus albus
-
alpha-D-glucose 1-phosphate + D-glucose
-
r

Organism

Organism UniProt Comment Textmining
Ruminococcus albus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-chelating column chromatography, and gel filtration Ruminococcus albus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
-
Ruminococcus albus ?
-
?
alpha-D-glucose 1-phosphate + D-glucosamine
-
Ruminococcus albus 4-O-beta-D-glucopyranosyl-D-glucosamine
-
?
alpha-D-glucose 1-phosphate + D-glucose
-
Ruminococcus albus cellobiose + phosphate
-
r
alpha-D-glucose 1-phosphate + D-mannose
-
Ruminococcus albus 4-O-beta-D-glucopyranosyl-D-mannose
-
?
alpha-D-glucose 1-phosphate + N-acetyl-D-glucosamine substrate for mutant Y648V only Ruminococcus albus 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine
-
?
cellobiose + phosphate
-
Ruminococcus albus alpha-D-glucose 1-phosphate + D-glucose
-
r

Synonyms

Synonyms Comment Organism
Cbp
-
Ruminococcus albus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
40
-
the enzyme is stable below 40°C Ruminococcus albus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.626
-
D-mannose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
1.3
-
alpha-D-glucose 1-phosphate mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
2.83
-
D-mannose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
5.27
-
D-mannose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
6.54
-
N-acetyl-D-glucosamine mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
7.83
-
2-deoxy-D-glucose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
9.56
-
D-glucosamine wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
21.5
-
2-deoxy-D-glucose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
35.4
-
D-glucosamine mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
58.3
-
D-mannose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
64.2
-
alpha-D-glucose 1-phosphate mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
73.9
-
cellobiose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
79.1
-
cellobiose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
88.9
-
cellobiose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
92.3
-
alpha-D-glucose 1-phosphate wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
96.5
-
alpha-D-glucose 1-phosphate mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
119
-
2-deoxy-D-glucose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
-
Ruminococcus albus

pH Stability

pH Stability pH Stability Maximum Comment Organism
5.5 8.5 the enzyme remains stable over the pH range of 5.5 to 8.5 Ruminococcus albus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0198
-
2-deoxy-D-glucose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
0.0263
-
D-mannose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
0.0376
-
N-acetyl-D-glucosamine mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
0.0477
-
2-deoxy-D-glucose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
0.0532
-
D-mannose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
0.0681
-
cellobiose mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
0.0829
-
alpha-D-glucose 1-phosphate mutant enzyme Y648V, at pH 6.0 and 37°C Ruminococcus albus
0.112
-
D-mannose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
0.357
-
2-deoxy-D-glucose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
0.61
-
D-glucosamine mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
0.719
-
D-glucosamine wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
0.931
-
D-mannose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
1.43
-
2-deoxy-D-glucose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
2.09
-
alpha-D-glucose 1-phosphate mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
2.36
-
cellobiose mutant enzyme Y648F, at pH 6.0 and 37°C Ruminococcus albus
5.35
-
cellobiose mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
8.55
-
alpha-D-glucose 1-phosphate mutant enzyme C485A, at pH 6.0 and 37°C Ruminococcus albus
47.1
-
cellobiose wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus
63.2
-
alpha-D-glucose 1-phosphate wild type enzyme, at pH 6.0 and 37°C Ruminococcus albus