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Literature summary for 2.4.2.12 extracted from

  • Burgos, E.S.; Schramm, V.L.
    Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase (2008), Biochemistry, 47, 11086-11096.
    View publication on PubMedView publication on EuropePMC

Activating Compound

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(Commentary)

Cloned (Comment) Organism
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys Homo sapiens

Inhibitors

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Homo sapiens P43490
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
phosphoprotein active phosphorylated NAMPT species neither isolated nor characterized Homo sapiens

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
Nampt
-
Homo sapiens
nicotinamide phosphoribosyltransferase
-
Homo sapiens

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
for both, NMN synthesis and ATP hydrolysis activity Homo sapiens

pH Range

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 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 [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