Cloned (Comment) | Organism |
---|---|
wild-type and mutants overexpressed from pFCT6.9 vector as His6-tagged fusion proteins in Escherichia coli BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D201N | activity is highly impaired, 0.2% of the specific activity of wild-type enzyme | Escherichia coli |
D201N/E390A | very similar specific specific activity to the wild-type | Escherichia coli |
D201N/Y313F | shows less than 0.1% of the specific activity present in the wild-type | Escherichia coli |
D201N/Y313F/E390A | shows less than 0.1% of the specific activity present in the wild-type | Escherichia coli |
E390A | specific activity is very similar to that of the wild-type | Escherichia coli |
E542A | extraordinary catalysis and binding abilities that turns RNase II into a super-enzyme. More than a 100fold increase in the specific exoribonucleolytic activity, significantly increases affinity for the poly(A) substrate | Escherichia coli |
R500A | shows more than a 40000fold reduction in specific activity when compared with the wild-type | Escherichia coli |
R500K | shows less than 0.1% of the specific activity present in the wild-type | Escherichia coli |
Y313A | 100fold reduction of specific activity | Escherichia coli |
Y313F | specific activity is very similar to that of the wild-type | Escherichia coli |
Y313F/E390A | specific activity is not affected | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0003 | - |
Poly(A) | 35-nt poly(A), mutant E542A, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli | |
0.00125 | - |
Poly(A) | 35-nt poly(A), wild-type, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required for the catalysis | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P30850 | - |
- |
Purification (Comment) | Organism |
---|---|
all mutants, with the exception of R500K, by histidine affinity chromatography and the AKTA fast protein liquid chromatography system | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | RNase II is only able to cleave DNA bases when having a ribose in the 2nd or the 4th positions | Escherichia coli | ? | - |
? | |
poly(A) + H2O | 35-nucleotide poly(A) chain, Tyr-313 and Glu-390 are crucial for RNA specificity of RNase II, Arg-500 residue in the active site is crucial for activity but not for RNA binding. Inside the cavity the unique specific contacts for ribose established by RNase II are those with the 2nd and 4th nucleotides from the 3'-end of the RNA molecule. These contacts are necessary and sufficient for cleavage to occur, and therefore, they seem to be responsible for the RNA specificity versus DNA in RNase II | Escherichia coli | 5'-AMP + oligonucleotide | - |
? |
Synonyms | Comment | Organism |
---|---|---|
RNase II | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.41 | - |
Poly(A) | 35-nt poly(A), wild-type, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli | |
80800 | - |
Poly(A) | 35-nt poly(A), mutant E542A, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
230 | - |
Poly(A) | 35-nt poly(A), wild-type, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli | |
236 | - |
Poly(A) | 35-nt poly(A), mutant E542A, at 37°C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol | Escherichia coli |