Cloned (Comment) | Organism |
---|---|
overexpression of wild-type and mutant enzyme | Escherichia coli |
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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0A7Y4 | type 1 enzyme | - |
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 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 | 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 | Comment | Organism |
---|---|---|
ribonuclease HI | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |