BRENDA - Enzyme Database show

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
EC Number
Activating Compound
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
diphosphate
enhanced ATPase activity
Homo sapiens
2.4.2.12
nicotinamide
enhanced ATPase activity
Homo sapiens
Cloned(Commentary)
EC Number
Commentary
Organism
2.4.2.12
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys
Homo sapiens
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
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
2.4.2.12
ATP
-
Homo sapiens
2.4.2.12
FK866
powerful, non-competitive inhibitor with and without ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
NAD+
competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide
Homo sapiens
2.4.2.12
NADH
competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide
Homo sapiens
2.4.2.12
nicotinamide
substrate inhibition
Homo sapiens
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.000855
-
nicotinamide
+/-0.000022 mM, pH 7.5, 100 microM PRPP, 88 nM NAMPT, HPLC-based quantification
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.18
-
phosphate
+/-0.02 mM, pH 7.5, 88 nM NAMPT, HPLC-based quantification
Homo sapiens
2.4.2.12
0.44
-
ADP
+/-0.05 mM, ADP-ATP radiolabel exchange in absence of other reactants
Homo sapiens
2.4.2.12
2.2
-
ATP
+/-0.2 mM, ADP-ATP radiolabel exchange in absence of other reactants
Homo sapiens
2.4.2.12
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)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.4.2.12
Homo sapiens
P43490
-
-
Posttranslational Modification
EC Number
Posttranslational Modification
Commentary
Organism
2.4.2.12
phosphoprotein
active phosphorylated NAMPT species neither isolated nor characterized
Homo sapiens
Purification (Commentary)
EC Number
Commentary
Organism
2.4.2.12
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)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.4.2.12
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
2.4.2.12
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
-
-
-
?
2.4.2.12
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
2.4.2.12
additional information
facultative ATP hydrolysis activity, not tightly coupled to NMN synthesis
690900
Homo sapiens
?
-
-
-
-
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
-
690900
Homo sapiens
nicotinamide mononucleotide + diphosphate
-
-
-
r
2.4.2.12
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
2.4.2.12
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
2.4.2.12
phospho-NAMPT + H2O
DELTAG° = -9.2 kcal/mol
690900
Homo sapiens
NAMPT + phosphate
-
-
-
?
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.4.2.12
0.0003
-
ATP
ATPase activity is decreased in simultaneous presence of 1 mM of substrates of forward and reverse NAMPT reaction
Homo sapiens
2.4.2.12
0.00137
-
nicotinamide
+/-0.00002, KM/kcat = 1600 1/M*s, pH 7.5, 100 microM PRPP, 88 nM NAMPT
Homo sapiens
2.4.2.12
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
2.4.2.12
0.00183
-
ATP
ATPase (ATP hydrolysis) activity of the free NAMPT, 2 mM ATP
Homo sapiens
2.4.2.12
0.00617
-
ATP
ATPase activity is enhanced in presence of 1 mM nicotinamide, 2 mM ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.4.2.12
7.5
-
for both, NMN synthesis and ATP hydrolysis activity
Homo sapiens
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
2.4.2.12
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
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
2.4.2.12
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]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.4.2.12
additional information
-
ATP
> 4 mM, inhibition of NMN synthesis by phosphorylated NAMPT
Homo sapiens
2.4.2.12
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
2.4.2.12
additional information
-
NAD+
> 100 microM, with respect to nicotinamide, in absence of ATP
Homo sapiens
2.4.2.12
additional information
-
NADH
> 100 microM, with respect to nicotinamide, in absence of ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.00025
-
nicotinamide
NMN synthesis
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.003
-
nicotinamide
NMN synthesis, in presence of 5 microM NAD+
Homo sapiens
2.4.2.12
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
2.4.2.12
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)
EC Number
Activating Compound
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
diphosphate
enhanced ATPase activity
Homo sapiens
2.4.2.12
nicotinamide
enhanced ATPase activity
Homo sapiens
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.4.2.12
chemically synthesised DNA (DNA 2.0) in pBAD DEST 49 for expression in Escherichia coli BL21 (DE3)-pLys
Homo sapiens
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
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
2.4.2.12
ATP
-
Homo sapiens
2.4.2.12
FK866
powerful, non-competitive inhibitor with and without ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
NAD+
competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide
Homo sapiens
2.4.2.12
NADH
competitive inhibitor of non-ATP and ATP coupled NMN synthesis with respect to PRPP and nicotinamide
Homo sapiens
2.4.2.12
nicotinamide
substrate inhibition
Homo sapiens
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.4.2.12
additional information
-
ATP
> 4 mM, inhibition of NMN synthesis by phosphorylated NAMPT
Homo sapiens
2.4.2.12
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
2.4.2.12
additional information
-
NAD+
> 100 microM, with respect to nicotinamide, in absence of ATP
Homo sapiens
2.4.2.12
additional information
-
NADH
> 100 microM, with respect to nicotinamide, in absence of ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.00025
-
nicotinamide
NMN synthesis
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.003
-
nicotinamide
NMN synthesis, in presence of 5 microM NAD+
Homo sapiens
2.4.2.12
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
2.4.2.12
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)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.000855
-
nicotinamide
+/-0.000022 mM, pH 7.5, 100 microM PRPP, 88 nM NAMPT, HPLC-based quantification
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
0.18
-
phosphate
+/-0.02 mM, pH 7.5, 88 nM NAMPT, HPLC-based quantification
Homo sapiens
2.4.2.12
0.44
-
ADP
+/-0.05 mM, ADP-ATP radiolabel exchange in absence of other reactants
Homo sapiens
2.4.2.12
2.2
-
ATP
+/-0.2 mM, ADP-ATP radiolabel exchange in absence of other reactants
Homo sapiens
2.4.2.12
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)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Posttranslational Modification (protein specific)
EC Number
Posttranslational Modification
Commentary
Organism
2.4.2.12
phosphoprotein
active phosphorylated NAMPT species neither isolated nor characterized
Homo sapiens
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.4.2.12
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)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.4.2.12
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
2.4.2.12
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
-
-
-
?
2.4.2.12
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
2.4.2.12
additional information
facultative ATP hydrolysis activity, not tightly coupled to NMN synthesis
690900
Homo sapiens
?
-
-
-
-
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl 1-diphosphate
-
690900
Homo sapiens
nicotinamide mononucleotide + diphosphate
-
-
-
r
2.4.2.12
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
2.4.2.12
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
2.4.2.12
phospho-NAMPT + H2O
DELTAG° = -9.2 kcal/mol
690900
Homo sapiens
NAMPT + phosphate
-
-
-
?
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.4.2.12
0.0003
-
ATP
ATPase activity is decreased in simultaneous presence of 1 mM of substrates of forward and reverse NAMPT reaction
Homo sapiens
2.4.2.12
0.00137
-
nicotinamide
+/-0.00002, KM/kcat = 1600 1/M*s, pH 7.5, 100 microM PRPP, 88 nM NAMPT
Homo sapiens
2.4.2.12
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
2.4.2.12
0.00183
-
ATP
ATPase (ATP hydrolysis) activity of the free NAMPT, 2 mM ATP
Homo sapiens
2.4.2.12
0.00617
-
ATP
ATPase activity is enhanced in presence of 1 mM nicotinamide, 2 mM ATP
Homo sapiens
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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
2.4.2.12
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)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.4.2.12
7.5
-
for both, NMN synthesis and ATP hydrolysis activity
Homo sapiens
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
2.4.2.12
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)
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
2.4.2.12
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