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Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase

Burgos, E.S.; Schramm, V.L.; Biochemistry 47, 11086-11096 (2008)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
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)
Commentary
Organism
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys
Homo sapiens
Inhibitors
Inhibitors
Commentary
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
Commentary
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
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Homo sapiens
P43490
-
-
Posttranslational Modification
Posttranslational Modification
Commentary
Organism
phosphoprotein
active phosphorylated NAMPT species neither isolated nor characterized
Homo sapiens
Purification (Commentary)
Commentary
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
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
690900
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
690900
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
690900
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
690900
Homo sapiens
?
-
-
-
-
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
-
690900
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
690900
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
690900
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
690900
Homo sapiens
NAMPT + phosphate
-
-
-
?
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
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
Commentary
Organism
7.5
-
for both, NMN synthesis and ATP hydrolysis activity
Homo sapiens
pH Range
pH Minimum
pH Maximum
Commentary
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
Commentary
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
Commentary
Organism
Structure
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; 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; 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; 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
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
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
Activating Compound (protein specific)
Activating Compound
Commentary
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) (protein specific)
Commentary
Organism
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys
Homo sapiens
Inhibitors (protein specific)
Inhibitors
Commentary
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
Ki Value [mM] (protein specific)
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
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; 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; 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; 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
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
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
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
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) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Posttranslational Modification (protein specific)
Posttranslational Modification
Commentary
Organism
phosphoprotein
active phosphorylated NAMPT species neither isolated nor characterized
Homo sapiens
Purification (Commentary) (protein specific)
Commentary
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) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
690900
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
690900
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
690900
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
690900
Homo sapiens
?
-
-
-
-
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
-
690900
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
690900
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
690900
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
690900
Homo sapiens
NAMPT + phosphate
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
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 (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
for both, NMN synthesis and ATP hydrolysis activity
Homo sapiens
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
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 (protein specific)
pH Stability
pH Stability Maximum
Commentary
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
Other publictions for EC 2.4.2.12
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
735765
Clark
Application of virtual screeni ...
Homo sapiens
Bioorg. Med. Chem. Lett.
26
2920-2926
2016
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-
-
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-
17
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1
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17
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1
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-
-
-
735766
Chen
Identification of benzothiophe ...
Homo sapiens
Bioorg. Med. Chem. Lett.
26
765-768
2016
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-
-
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-
17
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2
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14
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14
17
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1
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3
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1
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-
-
-
-
-
-
-
-
-
735872
Grolla
Extracellular nicotinamide pho ...
Homo sapiens
Br. J. Pharmacol.
173
2182-2194
2016
-
-
-
-
14
-
1
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1
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1
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49
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1
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14
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49
-
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1
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-
-
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-
2
1
1
2
-
-
736091
Fang
Analysis of the nicotinamide p ...
Danio rerio
FEBS J.
282
2858-2878
2015
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-
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1
1
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3
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1
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1
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1
-
-
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-
-
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-
1
1
-
-
-
736115
Marletta
Crystal structure of human nic ...
Homo sapiens
FEBS Open Bio
5
419-428
2015
-
-
-
-
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-
1
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1
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4
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1
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-
-
737247
Xu
Discovery and characterization ...
Homo sapiens
Sci. Rep.
5
10043
2015
-
-
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49
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1
1
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2
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4
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1
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48
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48
49
-
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1
1
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-
4
-
-
1
1
-
-
-
-
-
-
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-
-
-
-
-
-
-
737279
Zabka
Retinal toxicity, in vivo and ...
Homo sapiens, Rattus norvegicus
Toxicol. Sci.
144
163-172
2015
-
-
-
-
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-
14
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2
-
7
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6
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2
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14
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2
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6
-
-
2
-
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-
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-
-
-
-
-
-
-
-
-
735760
Zheng
Discovery of potent and effica ...
Homo sapiens
Bioorg. Med. Chem. Lett.
24
337-343
2014
-
-
-
-
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-
18
-
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1
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2
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1
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1
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14
-
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-
14
18
-
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1
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1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
736003
Hsu
The function of nicotinamide p ...
Homo sapiens
DNA Repair
23
64-68
2014
-
-
1
-
-
-
1
-
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-
1
1
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2
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1
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1
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1
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1
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1
1
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1
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-
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
736227
Pittelli
Nicotinamide phosphoribosyltra ...
Homo sapiens, Mus musculus
Immunol. Cell Biol.
92
191-199
2014
-
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-
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2
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2
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4
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2
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2
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2
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3
3
-
-
-
735758
Zheng
Identification of amides deriv ...
Homo sapiens
Bioorg. Med. Chem. Lett.
23
5488-5497
2013
-
-
-
-
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-
32
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1
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6
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6
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1
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28
-
-
-
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-
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28
32
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1
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6
-
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1
-
-
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-
-
-
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-
-
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-
-
736436
Tan
Pharmacological inhibition of ...
Homo sapiens
J. Biol. Chem.
288
3500-3511
2013
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12
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1
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1
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12
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1
-
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2
2
-
-
-
736612
Zheng
Structure-based discovery of n ...
Homo sapiens
J. Med. Chem.
56
6413-6433
2013
-
-
-
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-
40
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1
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2
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5
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1
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35
-
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-
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35
40
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-
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1
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5
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
723576
Bowlby
Nicotinamide phosphoribosyl tr ...
Homo sapiens
PLoS ONE
7
e40195
2012
-
-
-
-
-
-
-
-
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1
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5
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5
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
723588
Liu
Nicotinamide phosphoribosyltra ...
Mus musculus
PLoS ONE
7
e44933
2012
-
-
-
-
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1
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2
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1
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1
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-
721273
Zhang
A fluorometric assay for high- ...
Mus musculus
Anal. Biochem.
412
18-25
2011
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1
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4
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2
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1
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4
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1
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4
4
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1
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1
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-
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-
-
722099
Friebe
Leucocytes are a major source ...
Homo sapiens
Diabetologia
54
1200-1211
2011
-
-
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2
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2
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722182
Dahl
Nicotinamide phosphoribosyltra ...
Homo sapiens
Eur. J. Clin. Invest.
41
1098-1104
2011
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1
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1
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1
-
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1
-
-
722560
Zhang
Nicotinamide phosphoribosyltra ...
Homo sapiens
J. Bioanal. Biomed.
3
13-25
2011
-
-
1
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2
-
1
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1
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2
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1
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9
-
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1
1
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1
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2
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1
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1
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1
-
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9
-
-
1
1
-
-
-
-
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-
1
-
2
2
-
-
-
722718
Hong
Nicotinamide phosphoribosyltra ...
Oryctolagus cuniculus
J. Biol. Chem.
286
28619-28631
2011
-
-
-
-
-
-
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723535
Hara
Nicotinamide phosphoribosyltra ...
Homo sapiens
PLoS ONE
6
e22781
2011
1
-
1
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-
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-
4
-
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3
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7
-
1
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1
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1
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4
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7
-
1
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702085
Garten
Nicotinamide phosphoribosyltra ...
Homo sapiens
Biochem. Biophys. Res. Commun.
391
376-381
2010
-
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2
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3
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3
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3
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2
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-
704364
Takahashi
Structure and reaction mechani ...
Homo sapiens
J. Biochem.
147
95-107
2010
-
-
-
1
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2
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1
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1
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1
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705072
Colombano
A novel potent nicotinamide ph ...
Homo sapiens
J. Med. Chem.
53
616-623
2010
-
-
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1
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1
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1
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-
721934
Olesen
Target enzyme mutations are th ...
Homo sapiens
BMC Cancer
10
677
2010
-
-
1
-
5
-
3
-
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1
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1
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4
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1
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2
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1
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5
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2
3
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1
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4
-
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1
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-
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-
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722688
Pittelli
Inhibition of nicotinamide pho ...
Homo sapiens
J. Biol. Chem.
285
34106-34114
2010
-
-
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-
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1
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2
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2
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1
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2
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-
-
-
-
-
-
722862
Zhang
Nicotinamide phosphoribosyltra ...
Homo sapiens
J. Cell. Biochem.
110
1464-1470
2010
-
-
-
-
-
-
-
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1
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2
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1
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1
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1
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1
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