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Literature summary for 2.4.2.8 extracted from

  • Butterworth, A.C.; Medrano, F.J.; Eakin, A.E.; Craig, S.P., 3rd
    Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi (2004), Biochim. Biophys. Acta, 1699, 87-94.
    View publication on PubMed

Application

Application Comment Organism
pharmacology enzyme is a potential drug target in the treatment of parasite caused disease Trypanosoma cruzi

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli Trypanosoma cruzi

Protein Variants

Protein Variants Comment Organism
G69S reduced activity compared to the wild-type enzyme Trypanosoma cruzi
L67M highly reduced activity with hypoxanthine compared to the wild-type enzyme, mutant is not active with guanine Trypanosoma cruzi
additional information saturation mutagenesis and complement selection for investigation of functional roles of residues Leu67 and Gly69, sequencing of 70 clones and identification of 30 different mutations, several mutants of L67 or G69 support bacterial growth on minimal medium, but only L67M and G69S can be expressed and purified from overexpressing bacteria, overview Trypanosoma cruzi

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0086
-
hypoxanthine pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
0.012
-
guanine pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
0.0192
-
hypoxanthine pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.022
-
guanine pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.027
-
IMP pH 7.5, 37°C, recombinant wild-type enzyme and mutant L67M Trypanosoma cruzi
0.032
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
0.0339
-
hypoxanthine pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.035
-
diphosphate pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
0.08
-
IMP pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.083
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.132
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.135
-
diphosphate pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.214
-
diphosphate pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ at 12 mM Trypanosoma cruzi

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
GMP + diphosphate Trypanosoma cruzi
-
guanine + 5-phospho-alpha-D-ribose 1-diphosphate
-
r
IMP + diphosphate Trypanosoma cruzi
-
hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate
-
r

Organism

Organism UniProt Comment Textmining
Trypanosoma cruzi
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli by GMP affinity and cation exchange chromatography Trypanosoma cruzi

Reaction

Reaction Comment Organism Reaction ID
IMP + diphosphate = hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate reaction mechanism, function and influence of invariant active site loop I residues on enzyme efficiency in forward and reverse reaction Trypanosoma cruzi

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
-
Trypanosoma cruzi

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
GMP + diphosphate
-
Trypanosoma cruzi guanine + 5-phospho-alpha-D-ribose 1-diphosphate
-
r
IMP + diphosphate
-
Trypanosoma cruzi hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate
-
r
additional information L67 side chain participates in hydrophobic interactions between dimer subunits important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of 5-phospho-alpha-D-ribose 1-diphosphate, diphosphate, and hypoxanthine, residue 69 needs to be small like Gly, which is found there, to avoid sterical interference with binding of 5-phospho-alpha-D-ribose 1-diphosphate and diphosphate as well as the positioning of a metal ion Trypanosoma cruzi ?
-
?

Synonyms

Synonyms Comment Organism
HPRT
-
Trypanosoma cruzi

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Trypanosoma cruzi

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.07
-
IMP pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.12
-
diphosphate pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.19
-
IMP pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.3
-
hypoxanthine pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
0.31
-
diphosphate pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
0.47
-
IMP pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
0.48
-
diphosphate pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
4.7
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant mutant L67M Trypanosoma cruzi
20
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
22
-
hypoxanthine pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
22.1
-
guanine pH 7.5, 37°C, recombinant mutant G69S Trypanosoma cruzi
22.9
-
hypoxanthine pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
23.2
-
5-phospho-alpha-D-ribose 1-diphosphate pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi
32.9
-
guanine pH 7.5, 37°C, recombinant wild-type enzyme Trypanosoma cruzi

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Trypanosoma cruzi