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Literature summary for 2.4.2.8 extracted from

  • de Souza Dantas, D.; Ramos Dos Santos, C.; Guimaraes Pereira, G.A.; Medrano, F.J.
    Biochemical and structural characterization of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Pyrococcus horikoshii (2008), Biochim. Biophys. Acta, 1784, 953-960.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
from genomic DNA in pGEM-T-Eeasy, in pET15b for expression with hexa-His tag in Escherichia coli B21 Star (DE3) Pyrococcus horikoshii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information no significant change in KM with temperature (40-70°C) for all substrates tested Pyrococcus horikoshii
0.00326
-
guanine +/-0.00029 mM, 70°C Pyrococcus horikoshii
0.00388
-
hypoxanthine +/-0.00033 mM, 40°C Pyrococcus horikoshii
0.00483
-
hypoxanthine +/-0.00049 mM, 60°C Pyrococcus horikoshii
0.00485
-
guanine +/-0.00056 mM, 50°C Pyrococcus horikoshii
0.0061
-
hypoxanthine +/-0.00143 mM, 70°C Pyrococcus horikoshii
0.00702
-
hypoxanthine +/-0.00104 mM, 50°C Pyrococcus horikoshii
0.0282
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.0015 mM, 60°C Pyrococcus horikoshii
0.0369
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.0024 mM, 70°C Pyrococcus horikoshii
0.0412
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.0016 mM, 50°C Pyrococcus horikoshii
0.0428
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.0009 mM, 40°C Pyrococcus horikoshii
0.095
-
xanthine +/-0.0084 mM, 70°C Pyrococcus horikoshii
0.1012
-
xanthine +/-0.0101 mM, 50°C Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
103000
-
dynamic light scattering Pyrococcus horikoshii
103100
-
gel filtration chromatography Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Pyrococcus horikoshii
-
hyperthermophile
-

Purification (Commentary)

Purification (Comment) Organism
thermal treatment (1 h, 70°C), immobilized metal affinity chromatography with Hi-Trap nickel chelating column (pH 7.5, elution with 200 mM imidazole), cleavage of His-tag with thrombin followed by second IMAC Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
guanine + 5-phospho-alpha-D-ribose 1-diphosphate
-
Pyrococcus horikoshii GMP + diphosphate
-
?
guanine + 5-phospho-alpha-D-ribose 1-diphosphate hypoxanthine and guanine preferred substrates Pyrococcus horikoshii GMP + diphosphate
-
ir
hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate
-
Pyrococcus horikoshii IMP + diphosphate
-
?
hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate hypoxanthine and guanine preferred substrates, continuous spectrophotometric enzyme assay, pH 7.5, 12 mM MgCl2, 40-70°C, 30 sec Pyrococcus horikoshii IMP + diphosphate Kd(diphosphate): 4.7 +/-0.1 mM, revealed by quenching of intrinsic fluorescence of the enzyme upon diphosphate-binding ir
additional information no formation of PRPP and 6-oxopurine by pyrophosphorylysis due to disability of binding to nucleotides IMP, GMP, XMP at 40-70°C and substrate concentrations of up to 0.5 mM Pyrococcus horikoshii ?
-
?
xanthine + 5-phospho-alpha-D-ribose 1-diphosphate low catalytic efficiency (kcat/KM), xanthine is not preferred but a usable substrate Pyrococcus horikoshii XMP + diphosphate
-
ir

Subunits

Subunits Comment Organism
homohexamer dynamic light scattering (pH 7.5, 25 °C) and gel filtration chromatography (HR 10/30 Superdex 200, FPLC, pH 7.5) Pyrococcus horikoshii

Synonyms

Synonyms Comment Organism
6-oxopurine phosphoribosyltransferase
-
Pyrococcus horikoshii
HGXPRT
-
Pyrococcus horikoshii
HPRT
-
Pyrococcus horikoshii
hypoxanthine-guanine phosphoribosyltransferase
-
Pyrococcus horikoshii
hypoxanthine-guanine-xanthine phosphoribosyltransferase
-
Pyrococcus horikoshii
PhHGXPRT Pyrococcus horikoshii HGXPRT Pyrococcus horikoshii
PhPRT
-
Pyrococcus horikoshii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
additional information increasing kcat with temperature (40-70°C) for all substrates tested Pyrococcus horikoshii
additional information
-
additional information kcat/KM for 5-phospho-alpha-D-ribose 1-diphosphate = 0.00093 mM(-1)*min(-1) at 40°C, 0.00069 mM(-1)*min(-1) at 50°C, 0.00357 mM(-1)*min(-1) at 60°C, 0.00496 mM(-1)*min(-1) at 70°C Pyrococcus horikoshii
additional information
-
additional information kcat/KM for guanine = 0.00262 mM(-1)*min(-1) at 50°C, 0.02631 mM(-1)*min(-1) at 70°C Pyrococcus horikoshii
additional information
-
additional information kcat/KM for hypoxanthine = 0.00579 mM(-1)*min(-1) at 40°C, 0.00708 mM(-1)*min(-1) at 50°C, 0.01719 mM(-1)*min(-1) at 60°C, 0.03107 mM(-1)*min(-1) at 70°C Pyrococcus horikoshii
additional information
-
additional information kcat/KM for hypoxanthine similar to kcat/KM for guanine, kcat/KM for xanthine approximately 20 times lower Pyrococcus horikoshii
additional information
-
additional information kcat/KM for xanthine = 0.00040 mM(-1)*min(-1) at 50°C, 0.00139 mM(-1)*min(-1) at 70°C Pyrococcus horikoshii
0.21
-
guanine +/-0.01, 50°C Pyrococcus horikoshii
0.38
-
hypoxanthine +/-0.01, 40°C Pyrococcus horikoshii
0.48
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.02, 50°C Pyrococcus horikoshii
0.66
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.03, 40°C Pyrococcus horikoshii
0.68
-
xanthine +/-0.04, 50°C Pyrococcus horikoshii
0.83
-
hypoxanthine +/-0.04, 50°C Pyrococcus horikoshii
1.38
-
hypoxanthine +/-0.03, 60°C Pyrococcus horikoshii
1.43
-
guanine +/-0.02, 70°C Pyrococcus horikoshii
1.68
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.06, 60°C Pyrococcus horikoshii
2.2
-
xanthine +/-0.08, 70°C Pyrococcus horikoshii
3.05
-
5-phospho-alpha-D-ribose 1-diphosphate +/-0.07, 70°C Pyrococcus horikoshii
3.16
-
hypoxanthine +/-0.08, 70°C Pyrococcus horikoshii