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Literature summary for 3.2.2.1 extracted from

  • Vandemeulebroucke, A.; De Vos, S.; Van Holsbeke, E.; Steyaert, J.; Versees, W.
    A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax (2008), J. Biol. Chem., 283, 22272-22282.
    View publication on PubMed
Search for more literature for 3.2.2.1

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli WK6 cells Trypanosoma vivax

Crystallization (Commentary)

Crystallization (Comment) Organism
in complex with immucillin H, hanging drop vapour diffusion method, with 20% polyethylene glycol 4000 and 0.25 M ammonium sulfate in 10 mM potassium acetate, pH 5.0 Trypanosoma vivax

Protein Variants

Protein Variants Comment Organism
C248P mutant shows decreased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
D253P mutant shows increased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
E249P mutant shows decreased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
H247P mutant shows increased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
L250P mutant shows decreased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
L251P mutant shows increased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax
additional information the loop deletion mutant 3G hydrolyzes guanosine and 2-aminopurine riboside with a turnover comparable to that of the wild type, but an increase in Km is observed for both substrates, 7-methyl guanosine is hydrolyzed even faster by 3G compared with the wild type and hardly any difference is observed in the Km Trypanosoma vivax
R252P mutant shows increased kcat value compared to the wild type enzyme using guanosine as substrate Trypanosoma vivax

General Stability

General Stability Organism
once thawed, enzyme stocks are kept at 4°C and used over a period of several days but never refrozen to avoid activity loss Trypanosoma vivax

Inhibitors

Inhibitors Comment Organism Structure
immucillin H immucillin H is (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol Trypanosoma vivax

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0005
-
 7-methyl guanosine wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.0018
-
 guanosine mutant enzyme D253P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.0023
-
 guanosine wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.003
-
 guanosine mutant enzyme E249P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.004
-
 guanosine mutant enzyme C248P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.008
-
 guanosine mutant enzyme H247P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.009
-
 2-aminopurine riboside wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.016
-
 guanosine mutant enzyme L250P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.021
-
 guanosine mutant enzyme L251P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
0.024
-
 guanosine mutant enzyme R252P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax

Organism

Organism UniProt Comment Textmining
Trypanosoma vivax Q9GPQ4
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-NTA column chromatography and Superdex-75 gel filtration Trypanosoma vivax

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-aminopurine riboside + H2O
-
 Trypanosoma vivax 2-aminopurine + D-ribose
-
 ?
7-methyl guanosine + H2O
-
 Trypanosoma vivax D-ribose + 7-methyl guanine
-
 ?
adenosine + H2O
-
 Trypanosoma vivax D-ribose + adenine
-
 ?
guanosine + H2O
-
 Trypanosoma vivax D-ribose + guanine
-
 ?
inosine + H2O
-
 Trypanosoma vivax D-ribose + hypoxanthine
-
 ?

Synonyms

Synonyms Comment Organism
purine-specific nucleoside hydrolase
-
 Trypanosoma vivax

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.57
-
 2-aminopurine riboside wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
1.07
-
 7-methyl guanosine wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
1.09
-
 guanosine mutant enzyme C248P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
1.4
-
 guanosine mutant enzyme L250P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
1.7
-
 guanosine mutant enzyme E249P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
2.3
-
 guanosine wild type enzyme, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
2.5
-
 guanosine mutant enzyme L251P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
3.9
-
 guanosine mutant enzyme D253P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
4.1
-
 guanosine mutant enzyme H247P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax
13
-
 guanosine mutant enzyme R252P, in 50 mM potassium phosphate buffer, at 25°C and pH 7.0 Trypanosoma vivax