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Literature summary extracted from

  • Liu, H.; Woznica, K.; Catton, G.; Crawford, A.; Botting, N.; Naismith, J.H.
    Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from Homo sapiens (2007), J. Mol. Biol., 373, 755-763.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.2.19 in pEHISTEV (pBS-hQPRT as template) for expression in Escherichia coli BL21(DE3) or mutagenesis, expression with N-terminal hexa-His tag and TEV protease cleavage site, two extra N-terminal residues (Gly, Ala) remain after removal of hexa-His tag Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.2.19 PDB code: 2jbm (apo-hQPRTase), alpha/beta barrel fold (12 beta strands + 11 alpha helices) with N-terminal domain (residues 1-112, 279-291) and C-terminal domain (residues 113-278), similar to bacterial QPRTases (PDB: 1x1o), active site at alpha/beta open sandwich structure that faces an alpha/beta barrel of the adjacent subunit harbouring the quinolinic acid binding site (Arg102, Arg138, Arg161, Lys139, Lys171, pocket at the centre of the barrel), space group P2(1)2(1)2(1), unit-cell parameters: a = 111.5 A, b = 179.5 A, c = 194.7 A, 12 monomers in asymmetric unit arranged as 2 hexamers of D3 symmetry, sitting-drop vapour diffusion: 5 days, 20°C, 2 microlitre protein solution (10 mg/ml, pH7.5) + 2 microlitre precipitant (0.6 M potassium/sodium tartrate, 0.1 M sodium HEPES pH7.6), resolution of 2 A, phase determination using multiple wavelength anomalous diffraction on crystals of the Se-Met variant Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
2.4.2.19 K139A inactive, K139 is part of quinolinic acid binding site Homo sapiens
2.4.2.19 K139S inactive, K139 is part of quinolinic acid binding site Homo sapiens
2.4.2.19 K171A inactive, K171 is part of quinolinic acid binding site Homo sapiens
2.4.2.19 K171S inactive, K171 is part of quinolinic acid binding site Homo sapiens
2.4.2.19 R102A 10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer Homo sapiens
2.4.2.19 R102Q 10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer Homo sapiens
2.4.2.19 R138Q inactive, R138 is part of quinolinic acid binding site Homo sapiens
2.4.2.19 R161A 20% remaining activity, R161 is part of quinolinic acid binding site and important for substrate binding Homo sapiens
2.4.2.19 R161Q inactive, R161 is part of quinolinic acid binding site Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.4.2.19 5-phospho-alpha-D-ribose 1-diphosphate substrate inhibition, mixed inhibition (competitive and non-competitive) above 0.3 mM 5-phospho-alpha-D-ribose 1-diphosphate Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.2.19 0.0216
-
 quinolinic acid +/-0.003 mM, wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, affinity to quinolinic acid is independent of 5-phospho-alpha-D-ribose 1-diphosphate concentration Homo sapiens
2.4.2.19 0.0232
-
 5-phospho-alpha-D-ribose 1-diphosphate +/-0.0036 mM, wild-type, 0.3 mM quinolinic acid, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, KM for 5-phospho-alpha-D-ribose 1-diphosphate increases with decreasing quinolinic acid concentration Homo sapiens
2.4.2.19 0.319
-
 quinolinic acid +/-0.060 mM, mutant R161A, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C Homo sapiens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.4.2.19 Mg2+
-
 Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.2.19 pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate Homo sapiens type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis nicotinate D-ribonucleotide + diphosphate + CO2
-
 ir

Organism

EC Number Organism UniProt Comment Textmining
2.4.2.19 Homo sapiens Q15274
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.2.19 nickel affinity chromatography, TEV protease cleavage followed by nickel affinity chromatography, gel filtration, selenomethionine variant of hQPRTase purified in presence of 5 mM beta-mercaptoethanol Homo sapiens

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.4.2.19 additional information
-
 mutant R102A and R102Q, 10% remaining activity Homo sapiens
2.4.2.19 additional information
-
 mutant R161A, 20% remaining activity Homo sapiens
2.4.2.19 0.09
-
 wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, same order of magnitude as bacterial enzymes, 100% relative activity Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.2.19 additional information no detectable activity for mutants R161Q, R138Q, K171A, K171S, K139A, K139S, 6 mM MgCl2, pH7.2, 37°C, 0.3 mM quinolinic acid, UV-based assay: 14 microgram purified enzyme, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 30 min Homo sapiens ?
-
 ?
2.4.2.19 pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis Homo sapiens nicotinate D-ribonucleotide + diphosphate + CO2
-
 ir
2.4.2.19 quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate 6 mM MgCl2, pH7.2, 37°C, 0.3 mM quinolinic acid, HPLC-based assay: 7 microgram purified enzyme, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 20 min, UV-based assay: 14 microgram purified enzyme, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 30 min Homo sapiens nicotinic acid mononucleotide + diphosphate + CO2 HPLC-based assay: 254 nm (quantification of nicotinic acid mononucleotide and quinolinic acid), UV-based assay: 266 nm (quantification of nicotinic acid mononucleotide) ir

Subunits

EC Number Subunits Comment Organism
2.4.2.19 hexamer according to gel filtration, 3 canonical (AB) dimers (formed by two-fold rotation placing N-terminus of monomer A next to C-terminus of monomer B) related to each other by 3-fold rotation axis, hexameric D3 symmetry, active sites in close proximity at interface between monomers A and monomers B Homo sapiens

Synonyms

EC Number Synonyms Comment Organism
2.4.2.19 hQPRTase human QPRTase Homo sapiens
2.4.2.19 QPRTase
-
 Homo sapiens
2.4.2.19 quinolinate phosphoribosyltransferase
-
 Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.2.19 additional information
-
 quinolinic acid Vmax = 0.71(+/-0.0) microM/min, mutant R161A, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm Homo sapiens
2.4.2.19 additional information
-
 5-phospho-alpha-D-ribose 1-diphosphate Vmax = 0.93(+/-0.03) microM/min, wild-type, 0.3 mM quinolinic acid, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, Vmax increases with increasing 5-phospho-alpha-D-ribose 1-diphosphate concentrations up to 5 mM Homo sapiens
2.4.2.19 additional information
-
 quinolinic acid Vmax = 1.19(+/-0.05) microM/min, wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm Homo sapiens