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Literature summary for 3.1.26.4 extracted from

  • Tsunaka, Y.; Haruki, M.; Morikawa, M.; Oobatake, M.; Kanaya, S.
    Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI (2003), Biochemistry, 42, 3366-3374.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzyme Escherichia coli

Protein Variants

Protein Variants Comment Organism
D10A site-directed mutagenesis, active site mutant, poor binding of Mn2+ Escherichia coli
D10N site-directed mutagenesis, active site mutant, 1700fold increased dissociation constants for binding of Mn2+ compared to the wild-type enzyme Escherichia coli
D134A site-directed mutagenesis, mutant shows activity in presence of Mn2+, activity is similar to the wild-type enzyme, but the mutant is not inhibited by Mn2+ concentrations above 0.1 mM in contrast to the wild-type enzyme, 5.0fold increased dissociation constants for binding of Mn2+ Escherichia coli
D134N site-directed mutagenesis, mutant shows high activity in presence of Mn2+ without inhibition at higher Mn2+ concentrations, and 5.4fold increased dissociation constants for binding of Mn2+ Escherichia coli
D70N site-directed mutagenesis, active site mutant, 440fold increased dissociation constants for binding of Mn2+ compared to the wild-type enzyme Escherichia coli
E48A site-directed mutagenesis, mutant shows activity in presence of Mn2+, activity is similar to the wild-type enzyme, but the mutant is not inhibited by Mn2+ concentrations above 0.1 mM in contrast to the wild-type enzyme, 10fold increased dissociation constants for binding of Mn2+ Escherichia coli
E48A/D134A site-directed mutagenesis, active site mutant, highly reduced activity and 65fold increased dissociation constants for binding of Mn2+ compared to the wild-type enzyme Escherichia coli
E48A/D134N site-directed mutagenesis, active site mutant, reduced activity and 260fold increased dissociation constants for binding of Mn2+ compared to the wild-type enzyme Escherichia coli
E48Q site-directed mutagenesis, mutant shows activity in presence of Mn2+ and 9.2fold increased dissociation constants for binding of Mn2+ compared to the wild-type enzyme Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
Mn2+ wild-type enzyme, above 0.1 mM, activating below, activating metal ion binding site is site 1, inhibitory binding site is site 2 Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ maximal activity at 5 mM, binds to metal ion binding site 1 not 2, required, can substitute for Mn2+ Escherichia coli
Mn2+ required, maximal activity at 0.002-0.005 mM, can substitute for Mg2+, activates up to 0.1 mM, inhibitory above, enzyme contains 2 metal ion binding sites 1 and 2 with regulatory influence on each other, activating metal ion binding site is site 1, inhibitory binding site is site 2, overview, mutants E48A, E48Q, D134A, and D134N have only 1 active Mn2+-binding site Escherichia coli
additional information metal ion binding sites are located in the active site Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P0A7Y4 type 1 enzyme
-

Reaction

Reaction Comment Organism Reaction ID
Endonucleolytic cleavage to a 5'-phosphomonoester Asp10, Glu48, Asp70, and Asp134 are involved in catalysis, role of Mn2+ in catalysis, mechanism Escherichia coli

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.1
-
purified mutantE48A, at 10 mM MnCl2 Escherichia coli
1.5
-
purified mutant E48A/D134N at 10 mM MnCl2 Escherichia coli
4.7
-
purified wild-type enzyme, at 1 mM MnCl2 Escherichia coli
4.9
-
purified wild-type enzyme, at 10 mM MnCl2 Escherichia coli
15.2
-
purified wild-type enzyme, at 0.0005 mM MnCl2 Escherichia coli
24.2
-
purified mutant D134N, at 10 mM MnCl2 Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
RNA*DNA hybrid + H2O 12 bp and 29 bp oligomers, cleavage site specificity depends on bound metal ion, wild-type end mutant enzymes Escherichia coli ?
-
?

Synonyms

Synonyms Comment Organism
ribonuclease HI
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Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli