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Literature summary for 3.2.2.8 extracted from

  • Iovane, E.; Giabbai, B.; Muzzolini, L.; Matafora, V.; Fornili, A.; Minici, C.; Giannese, F.; Degano, M.
    Structural basis for substrate specificity in group I nucleoside hydrolases (2008), Biochemistry, 47, 4418-4426.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapour diffusion method, using 100 mM Tris (pH 8.5), 200 mM NaCl, and 24% PEG 4000 Escherichia coli

Protein Variants

Protein Variants Comment Organism
Q227A the mutation causes an increase of kcat for uridine and inosine Escherichia coli
Q227F the mutation causes an increase of kcat for uridine and inosine Escherichia coli
Q227Y the mutation has a strong, enhancing effect on the hydrolysis of inosine, and the catalytic efficiency for the purinic substrate is increased by a factor of 7.6 Escherichia coli
T223A the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine Escherichia coli
T223F the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine Escherichia coli
T223F/Q227Y the mutant shows a 2fold increase in catalytic efficiency toward inosine Escherichia coli
T223Y the mutation does not affect the specificity of the enzyme toward inosine or uridine Escherichia coli
T223Y/Q227Y the mutant displays a catalytic efficiency toward inosine that is more than 50fold increased compared to that of wild type enzyme Escherichia coli
T227A the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine Escherichia coli
T227F the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.12
-
uridine wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.31
-
uridine mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.33
-
uridine mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.57
-
uridine mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.77
-
uridine mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.06
-
uridine mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.09
-
uridine mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.13
-
uridine mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.16
-
Inosine mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.19
-
uridine mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.77
-
Inosine mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
1.93
-
Inosine mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
2.14
-
Inosine mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
2.34
-
Inosine wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
3.29
-
Inosine mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
4.31
-
Inosine mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
4.42
-
Inosine mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
5.3
-
Inosine mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ the substrate binds to the Ca2+-containing active site in the catalytic cavity at the C-terminal end of the core beta-sheet Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P33022
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-NTA column chromatography and MonoQ column chromatography Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
inosine + H2O poor substrate Escherichia coli hypoxanthine + D-ribose
-
?
uridine + H2O
-
Escherichia coli uracil + D-ribose
-
?

Synonyms

Synonyms Comment Organism
CU-NH
-
Escherichia coli
cytidine-uridine-preferring nucleoside hydrolase
-
Escherichia coli
RihB
-
Escherichia coli
YeiK
-
Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.035
-
Inosine mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.086
-
Inosine wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.109
-
Inosine mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.125
-
Inosine mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.18
-
Inosine mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.182
-
Inosine mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.382
-
Inosine mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
0.593
-
Inosine mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
3.62
-
Inosine mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
5.4
-
uridine wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
15.1
-
uridine mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
18.8
-
uridine mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
39.8
-
uridine mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
44.3
-
uridine mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
46.9
-
uridine mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
52.5
-
uridine mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
59.1
-
uridine mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli
72.9
-
uridine mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C Escherichia coli