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Literature summary for 4.2.2.8 extracted from

  • Pojasek, K.; Shriver, Z.; Hu, Y.; Sasisekharan, R.
    Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III (2000), Biochemistry, 39, 4012-4019.
    View publication on PubMed
Search for more literature for 4.2.2.8

Cloned(Commentary)

Cloned (Comment) Organism
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli, recombinant wild-type enzyme shows a lower activity than the native one Pedobacter heparinus

Protein Variants

Protein Variants Comment Organism
H105A PCR overlap extension site-directed mutagenesis, very low expression level, no measurement of activity possible, reduced expression level Pedobacter heparinus
H110A PCR overlap extension site-directed mutagenesis, reduced kcat, highly reduced Km compared to both recombinant and native wild-type enzymes, reduced expression level Pedobacter heparinus
H139A PCR overlap extension site-directed mutagenesis, reduced kcat and increased Km compared to both recombinant and native wild-type enzymes, reduced expression level Pedobacter heparinus
H152A PCR overlap extension site-directed mutagenesis, reduced Km and a kcat value between the recombinant and the native wild-type enzyme Pedobacter heparinus
H225A PCR overlap extension site-directed mutagenesis, Km is the same as for the recombinant wild-type, reduced kcat Pedobacter heparinus
H234A PCR overlap extension site-directed mutagenesis, Km is similar to the recombinant wild-type, reduced kcat Pedobacter heparinus
H241A PCR overlap extension site-directed mutagenesis, highly reduced kcat, highly reduced Km compared to both recombinant and native wild-type enzymes Pedobacter heparinus
H295A PCR overlap extension site-directed mutagenesis, inactive mutant Pedobacter heparinus
H36A PCR overlap extension site-directed mutagenesis, reduced Km and a kcat value between the recombinant and the native wild-type enzyme Pedobacter heparinus
H424A PCR overlap extension site-directed mutagenesis, reduced Km and kcat Pedobacter heparinus
H469A PCR overlap extension site-directed mutagenesis, reduced Km and increased kcat compared to both recombinant and native wild-type enzymes Pedobacter heparinus
H510A PCR overlap extension site-directed mutagenesis, inactive mutant Pedobacter heparinus
H539A PCR overlap extension site-directed mutagenesis, Km between the recombinant and the native wild-type enzyme, kcat is increased compared to both wild-type enzymes Pedobacter heparinus

Inhibitors

Inhibitors Comment Organism Structure
diethyl dicarbonate inactivation, 80% reversible by hydroxylamine within 6 h, mapping of modified histidine residues Pedobacter heparinus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.009
-
 heparan sulfate mutant H110A, pH 7.6, 35°C Pedobacter heparinus
0.016
-
 heparan sulfate mutant H241A, pH 7.6, 35°C Pedobacter heparinus
0.058
-
 heparan sulfate mutant H152A, pH 7.6, 35°C Pedobacter heparinus
0.059
-
 heparan sulfate mutant H424A, pH 7.6, 35°C Pedobacter heparinus
0.071
-
 heparan sulfate mutant H469A, pH 7.6, 35°C Pedobacter heparinus
0.075
-
 heparan sulfate mutant H234A, pH 7.6, 35°C Pedobacter heparinus
0.08
-
 heparan sulfate recombinant wild-type enzyme and mutant H225A, pH 7.6, 35°C Pedobacter heparinus
0.092
-
 heparan sulfate mutant H539A, pH 7.6, 35°C Pedobacter heparinus
0.098
-
 heparan sulfate mutant H36A, pH 7.6, 35°C Pedobacter heparinus
0.143
-
 heparan sulfate native wild-type enzyme, pH 7.6, 35°C Pedobacter heparinus
0.191
-
 heparan sulfate mutant H139A, pH 7.6, 35°C Pedobacter heparinus

Organism

Organism UniProt Comment Textmining
Pedobacter heparinus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutants from Escherichia coli Pedobacter heparinus

Reaction

Reaction Comment Organism Reaction ID
[GlcAbeta(1-4)GlcNbeta(1-4)]n = [GlcAbeta(1-4)GlcNbeta(1-4)]n-1 + 2-amino-2-deoxy-4-O-alpha-L-threo-hex-4-enopyranuronosyl-beta-D-glucopyranose His295 and His510 are essential for catalytic activity Pedobacter heparinus
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
heparan sulfate
-
 Pedobacter heparinus ?
-
 ?

Synonyms

Synonyms Comment Organism
heparinase III
-
 Pedobacter heparinus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
35
-
 assay at Pedobacter heparinus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5
-
 heparan sulfate mutant H241A, pH 7.6, 35°C Pedobacter heparinus
22
-
 heparan sulfate mutant H225A, pH 7.6, 35°C Pedobacter heparinus
23
-
 heparan sulfate mutant H234A, pH 7.6, 35°C Pedobacter heparinus
24
-
 heparan sulfate mutant H424A, pH 7.6, 35°C Pedobacter heparinus
37
-
 heparan sulfate mutant H110A, pH 7.6, 35°C Pedobacter heparinus
68
-
 heparan sulfate mutant H139A, pH 7.6, 35°C Pedobacter heparinus
78
-
 heparan sulfate recombinant wild-type enzyme, pH 7.6, 35°C Pedobacter heparinus
83
-
 heparan sulfate mutant H152A, pH 7.6, 35°C Pedobacter heparinus
86
-
 heparan sulfate mutant H36A, pH 7.6, 35°C Pedobacter heparinus
94
-
 heparan sulfate native wild-type enzyme, pH 7.6, 35°C Pedobacter heparinus
100
-
 heparan sulfate mutant H469A, pH 7.6, 35°C Pedobacter heparinus
132
-
 heparan sulfate mutant H539A, pH 7.6, 35°C Pedobacter heparinus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
 assay at Pedobacter heparinus

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
additional information
-
 additional information inactivation kinetics Pedobacter heparinus