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Literature summary for 3.1.13.1 extracted from

  • Barbas, A.; Matos, R.G.; Amblar, M.; Lopez-Vinas, E.; Gomez-Puertas, P.; Arraiano, C.M.
    Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME" (2009), J. Biol. Chem., 284, 20486-20498.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.13.1

Cloned(Commentary)

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required for the catalysis Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P30850
-
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
RNase II
-
 Escherichia coli

Turnover Number [1/s]

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 [mM/s]

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