Activating Compound | Comment | Organism | Structure |
---|---|---|---|
ATP | 35-fold higher concentrations of NMN synthesised, impact on substrate affinities, coupling of ATP hydrolysis to NMN synthesis enhances catalytic efficiency, accelerates forward turnover and overcomes the unfavourable thermodynamic equilibrium | Homo sapiens | |
diphosphate | enhanced ATPase activity | Homo sapiens | |
nicotinamide | enhanced ATPase activity | Homo sapiens |
Cloned (Comment) | Organism |
---|---|
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys | Homo sapiens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
5P-DADMe-NMN | poor, competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide, 3-fold increased binding in presence of ATP | Homo sapiens | |
alpha-D-5-phosphoribosyl 1-diphosphate | inhibits ATPase activity at 1 mM concentration, nearly complete inhibition of ADP-ATP exchange at 100 microM | Homo sapiens | |
ATP | - |
Homo sapiens | |
FK866 | powerful, non-competitive inhibitor with and without ATP | Homo sapiens | |
additional information | no impact on ADP-ATP exchange rate by 10 microM adenylate kinase inhibitor P(1), P(5)-di(adenosine-5-)pentaphosphate (Ap5A), NAMPT does not accommodate Ap5A | Homo sapiens | |
NAD+ | competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide | Homo sapiens | |
NADH | competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide | Homo sapiens | |
nicotinamide | substrate inhibition | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000005 | - |
nicotinamide | +/-0.000002 mM, in presence of 2 mM ATP the Km is 150-fold lowered, calculated from apparent KM by extrapolation to correct for inhibition by NAD+ in the continuous fluorimetric assay, pH 7.5, 200 microM PRPP, 2.8 nM NAMPT | Homo sapiens | |
0.000235 | - |
nicotinamide | +/-0.000017 mM, in presence of 2 mM phosphate, pH 7.5, 100 microM PRPP, 88 nM NAMPT, HPLC-based quantification | Homo sapiens | |
0.00063 | - |
alpha-D-5-phosphoribosyl 1-diphosphate | +/-0.00003 mM, in presence of 2 mM ATP the Km is lowered 10-fold, calculated from apparent KM by extrapolation to correct for inhibition by NAD+ in the continuous fluorimetric assay, pH 7.5, 125 nM nicotinamide, 2.2 nM NAMPT | Homo sapiens | |
0.000855 | - |
nicotinamide | +/-0.000022 mM, pH 7.5, 100 microM PRPP, 88 nM NAMPT, HPLC-based quantification | Homo sapiens | |
0.0072 | - |
alpha-D-5-phosphoribosyl 1-diphosphate | +/-0.0006 mM, pH 7.5, 1 microM nicotinamide, 88 nM NAMPT, HPLC-based quantification | Homo sapiens | |
0.0117 | - |
alpha-D-5-phosphoribosyl 1-diphosphate | +/-0.0008 mM, in presence of 2 mM phosphate, pH 7.5, 1 microM nicotinamide, 88 nM NAMPT, HPLC-based quantification | Homo sapiens | |
0.18 | - |
phosphate | +/-0.02 mM, pH 7.5, 88 nM NAMPT, HPLC-based quantification | Homo sapiens | |
0.44 | - |
ADP | +/-0.05 mM, ADP-ATP radiolabel exchange in absence of other reactants | Homo sapiens | |
2.2 | - |
ATP | +/-0.2 mM, ADP-ATP radiolabel exchange in absence of other reactants | Homo sapiens | |
7.4 | - |
ATP | +/-1.5 mM, in presence of 2 mM phosphate, pH 7.5, 100 nM nicotinamide, 50 microM PRPP, 2.2 nM NAMPT | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate | Homo sapiens | - |
nicotinamide mononucleotide + diphosphate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P43490 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
phosphoprotein | active phosphorylated NAMPT species neither isolated nor characterized | Homo sapiens |
Purification (Comment) | Organism |
---|---|
lysis of bacteria by French press, elution from Ni-NTA column by 100 and 200 mM imidazole, concentration and desalting, elution from HiLoad Superdex 200GP 26/60 column with 100 mM HEPES pH 7.5, 100 mM NaCl, 10 mM beta-mercaptoethanol, concentrated to 50-75 mg/ml, kept frozen | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ADP + ATP | isotope exchange reaction (in presence of radiolabeled ADP and ATP but in absence of other reactants, meaning without NMN synthesis) requires high-energy phosphorylated NAMPT | Homo sapiens | ATP + ADP | - |
r | |
ATP + H2O | DELTAG° = -7.3, in presence of NAMPT slow steady-state reaction after initial burst, ATPase activity in absence of other substrates, but can be modulated by addition of single substrates and mixtures | Homo sapiens | ADP + phosphate | - |
? | |
ATP + NAMPT | autophosphorylation (DELTAG° = 1.9 kcal/mol, Keq = 0.047) is unfavourable and appears as initial burst of ADP generation | Homo sapiens | ADP + phospho-NAMPT | at 2.5 mM ATP and 46 microM NAMPT 77% of NAMPT is phosphorylated (proposed: His247 phospho-NAMPT), hydrolysis of phospho-NAMPT at 0.8/min, active phosphorylated NAMPT species neither isolated nor characterized yet | r | |
additional information | facultative ATP hydrolysis activity, not tightly coupled to NMN synthesis | Homo sapiens | ? | - |
? | |
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate | - |
Homo sapiens | nicotinamide mononucleotide + diphosphate | - |
r | |
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate | DELTAG° = 1.1 kcal/mol, Keq = 0.15, unfavourable product-to-substrate ratio | Homo sapiens | nicotinamide mononucleotide + diphosphate | equilibrium was established after 3 h in presence of 100 microM PRPP, 100 microM diphosphate, 5 microM nicotinamide mononucleotide, 0.6 microM NAMPT | r | |
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate + ATP + H2O | DELTAG° = -1.0 kcal/mol, Keq = 5.0, reversion of the preestablished equilibrium in a non-ATP-coupled reaction, thermodynamic switch of the product-to-substrate ratio towards NMN production compared to the non-ATP-coupled reaction with an energy difference of -2.1 kcal/mol (meaning only 2.1 kcal/mol of 7.3 kcal/mol of the energy from ATP hydrolysis coupled to NMN formation), formation or hydrolysis of high-energy phosphorylated NAMPT may be rate limiting for overall ATP-coupled NMN synthesis | Homo sapiens | nicotinamide mononucleotide + diphosphate + ADP + phosphate | in presence of 2-2.5 mM ATP: 1 mol ADP per NMN formed (stoichiometric coupling ratio, R = 1), under optimal conditions (2 microM nicotinamide, 100 microM PRPP, 2.5 mM ATP, 5 mM Mg2+): R increases linearly with increasing diphosphate concentration | r | |
phospho-NAMPT + H2O | DELTAG° = -9.2 kcal/mol | Homo sapiens | NAMPT + phosphate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Nampt | - |
Homo sapiens |
nicotinamide phosphoribosyltransferase | - |
Homo sapiens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0003 | - |
ATP | ATPase activity is decreased in simultaneous presence of 1 mM of substrates of forward and reverse NAMPT reaction | Homo sapiens | |
0.00137 | - |
nicotinamide | +/-0.00002, KM/kcat = 1600 1/M*s, pH 7.5, 100 microM PRPP, 88 nM NAMPT | Homo sapiens | |
0.00142 | - |
nicotinamide | +/-0.00002, in presence of 2 mM phosphate, KM/kcat = 6100 1/M*s, pH 7.5, 100 microM PRPP, 88 nM NAMPT | Homo sapiens | |
0.00183 | - |
ATP | ATPase (ATP hydrolysis) activity of the free NAMPT, 2 mM ATP | Homo sapiens | |
0.00617 | - |
ATP | ATPase activity is enhanced in presence of 1 mM nicotinamide, 2 mM ATP | Homo sapiens | |
0.00767 | - |
nicotinamide | +/-0.00150, in presence of 2 mM ATP the catalytic efficiency (KM/kcat = 1800000 1/M*s) is improved 1100-fold, pH 7.5, 100 microM PRPP, 88 nM NAMPT | Homo sapiens | |
0.015 | - |
ATP | ADP-ATP radiolabel exchange in absence of other reactants (without NMN synthesis), formation or hydrolysis of phospho-NAMPT may be rate limiting for overall NMN synthesis reaction in presence of ATP | Homo sapiens | |
0.0183 | - |
ATP | ATPase activity is enhanced in presence of 1 mM diphosphate, presence of PRPP and/or nicotinamide reverses stimulation by diphosphate, 2 mM ATP | Homo sapiens | |
0.0433 | - |
ATP | ATPase activity in presence of 1 mM imidodiphosphate (PNP) is higher than in presence of pyophosphate, 2 mM ATP | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
for both, NMN synthesis and ATP hydrolysis activity | Homo sapiens |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
6.5 | 8.5 | preincubation of NAMPT for 1 h at 25 °C and various pH values in presence of 1 microM nicotinamide, 1 mM ATP, 30 nM NAMPT (NAMPT reaction started with 200 microM alpha-D-5-phosphoribosyl 1-diphosphate) or 2 mM ATP, 0.75 microM NAMPT (ATP hydrolysis) | Homo sapiens |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
6.5 | 8.5 | preincubation of 30 nM NAMPT for 1 h at 25 °C and various pH values, NMN synthesis: reaction started with 200 microM alpha-D-5-phosphoribosyl 1-diphosphate in presence of 1 microM nicotinamide, 1 mM ATP | Homo sapiens |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
NAD+ | > 100 microM, with respect to nicotinamide, in absence of ATP | Homo sapiens | |
additional information | - |
NADH | > 100 microM, with respect to nicotinamide, in absence of ATP | Homo sapiens | |
additional information | - |
ATP | > 4 mM, inhibition of NMN synthesis by phosphorylated NAMPT | Homo sapiens | |
additional information | - |
FK866 | 10 +/-1 pM, with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in presence of 2.5 mM ATP, KM/Ki = 63000, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
additional information | - |
FK866 | 150 +/-18 pM, with respect to nicotinamide, in presence of 2.5 mM ATP, KM/Ki = 33, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
additional information | - |
FK866 | 5000 +/-500 pM, with respect to nicotinamide, in absence of ATP, KM/Ki = 171, 100 microM PRPP, 1 microM nicotinamide, 88 nM NAMPT, 15 min | Homo sapiens | |
additional information | - |
FK866 | 730 +/-75 pM, with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in absence of ATP, KM/Ki = 9863, 100 microM PRPP, 1 microM nicotinamide, 88 nM NAMPT, 15 min | Homo sapiens | |
0.00014 | - |
NAD+ | with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in presence of 2.5 mM ATP, KM/Ki = 5, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.00016 | - |
5P-DADMe-NMN | with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in presence of 2.5 mM ATP, KM/Ki = 4, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.00022 | - |
NADH | with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in presence of 2.5 mM ATP, KM/Ki = 3, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.00025 | - |
nicotinamide | NMN synthesis | Homo sapiens | |
0.00053 | - |
5P-DADMe-NMN | with respect to alpha-D-5-phosphoribosyl 1-diphosphate (PRPP), in absence of ATP, KM/Ki = 14, 100 microM PRPP, 1 microM nicotinamide, 88 nM NAMPT, 15 min | Homo sapiens | |
0.0021 | - |
NAD+ | with respect to nicotinamide, in presence of 2.5 mM ATP, KM/Ki <1, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.0025 | - |
5P-DADMe-NMN | with respect to nicotinamide, in presence of 2.5 mM ATP, KM/Ki <1, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.003 | - |
nicotinamide | NMN synthesis, in presence of 5 microM NAD+ | Homo sapiens | |
0.0032 | - |
NADH | with respect to nicotinamide, in presence of 2.5 mM ATP, KM/Ki <1, 200 microM PRPP, 100 nM nicotinamide, 2.2 nM NAMPT, 15 min | Homo sapiens | |
0.0071 | - |
5P-DADMe-NMN | with respect to nicotinamide, in absence of ATP, KM/Ki <1, 100 microM PRPP, 1 microM nicotinamide, 88 nM NAMPT, 15 min | Homo sapiens |