BRENDA - Enzyme Database show

Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues

Kuhn, I.; Kellenberger, E.; Cakir-Kiefer, C.; Muller-Steffner, H.; Schuber, F.; Biochim. Biophys. Acta 1844, 1317-1331 (2014)

Data extracted from this reference:

Engineering
EC Number
Amino acid exchange
Commentary
Organism
3.2.2.6
D147A
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E138A
site-directed mutagenesis, the mutation causes a modest increase in the rate of NAD+ transformation which is proportional to its concentration. At 4.0 M, the rate increase is about 1.2fold and the formation of beta-1'-O-methyl ADP-ribose amounts to about 80% of the total reaction products. The observed selectivity in favor of methanolysis is similar to that of wild-type enzyme. The ADP-ribosyl cyclase activity of E138A mutant is more affected by the competing nucleophile, i.e. formation of ADP-ribose and cADPR are reduced by 75% and 90% respectively at 4.0 M methanol, the mutant shows an increase in ADP cyclization and higly reduced overall activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E138Q
site-directed mutagenesis, in the presence of methanol, mutant E138Q efficiently catalyzes the formation of beta-1'-O-methyl ADP-ribose. But in contrast with mutant E138A, and like the wild-type enzyme, solvolysis does not affect the overall turnover rate of NAD+ indicating that the formation of the E.ADP-ribosyl intermediate is still rate limiting
Bos taurus
3.2.2.6
E218A
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E218Q
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118A/W181A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate which is 16fold lower than the product of the effects of the two single mutations
Bos taurus
3.2.2.6
W118F
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118H
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W181A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate and a reduced sensitivity to nicotinamide inhibition compared to the wild-type enzyme
Bos taurus
3.2.2.6
W181F
site-directed mutagenesis, the mutant shows a decrease in activity and an increase in ADP cyclization compared to the wild-type enzyme
Bos taurus
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
3.2.2.6
1,N6-etheno NAD+
-
Bos taurus
3.2.2.6
2'-deoxy-2'-beta-D-fluoroarabinofuranoside NAD+
-
Bos taurus
3.2.2.6
2'-deoxy-2'-beta-D-fluororibofuranoside NAD+
non-covalent complex of the inhibitor formed with enzyme mutant E218Q, PDB ID: 3ghh, and with wild-type enzyme, PDB ID: 3kou
Bos taurus
3.2.2.6
3-aminopyridine
-
Bos taurus
3.2.2.6
Isonicotinic acid hydrazide
-
Bos taurus
3.2.2.6
nicotinamide
inhibition involves enzyme residue Trp181
Bos taurus
3.2.2.6
Pyridine
non-competitive inhibition of mutant E218A by pyridine
Bos taurus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.0171
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0175
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0189
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0215
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0247
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0276
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.028
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0289
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0295
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0297
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.036
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0453
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.2.2.6
NAD+ + H2O
Bos taurus
bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism
ADP-D-ribose + nicotinamide
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.2.2.6
Bos taurus
Q9TTF5
-
-
Reaction
EC Number
Reaction
Commentary
Organism
3.2.2.6
NAD+ + H2O = ADP-D-ribose + nicotinamide
substrate binding with a crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD+, stepwise ordered uni-bi kinetic mechanism, overview. Residues Trp118, Glu138, Asp147, Trp181 stabilize the ribooxocarbenium ion-like transition state mostly by electrostatic interactions
Bos taurus
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.2.2.6
additional information
selectivity in favor of methanolysis by wild-type enzyme and mutant E138A. 1'-Azido ADP-ribose is the reaction product obtained in the presence of azide. the ADP-ribosyl cyclase activity of wild-type bCD38 isminimal
731357
Bos taurus
?
-
-
-
-
3.2.2.6
NAD+ + H2O
bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism
731357
Bos taurus
ADP-D-ribose + nicotinamide
-
-
-
?
3.2.2.6
NAD+ + H2O
via a stepwise reaction mechanism
731357
Bos taurus
ADP-D-ribose + nicotinamide
-
-
-
?
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.2.2.6
37
-
assay at
Bos taurus
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.0155
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.016
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.03
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.122
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.49
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.81
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
3.5
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
6.64
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
7.8
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
10.36
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
18.37
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
57.9
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.2.2.6
7.4
-
assay at
Bos taurus
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
3.2.2.6
D147A
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E138A
site-directed mutagenesis, the mutation causes a modest increase in the rate of NAD+ transformation which is proportional to its concentration. At 4.0 M, the rate increase is about 1.2fold and the formation of beta-1'-O-methyl ADP-ribose amounts to about 80% of the total reaction products. The observed selectivity in favor of methanolysis is similar to that of wild-type enzyme. The ADP-ribosyl cyclase activity of E138A mutant is more affected by the competing nucleophile, i.e. formation of ADP-ribose and cADPR are reduced by 75% and 90% respectively at 4.0 M methanol, the mutant shows an increase in ADP cyclization and higly reduced overall activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E138Q
site-directed mutagenesis, in the presence of methanol, mutant E138Q efficiently catalyzes the formation of beta-1'-O-methyl ADP-ribose. But in contrast with mutant E138A, and like the wild-type enzyme, solvolysis does not affect the overall turnover rate of NAD+ indicating that the formation of the E.ADP-ribosyl intermediate is still rate limiting
Bos taurus
3.2.2.6
E218A
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
E218Q
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118A/W181A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate which is 16fold lower than the product of the effects of the two single mutations
Bos taurus
3.2.2.6
W118F
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W118H
site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme
Bos taurus
3.2.2.6
W181A
site-directed mutagenesis, the mutant shows a decrease of the catalytic rate and a reduced sensitivity to nicotinamide inhibition compared to the wild-type enzyme
Bos taurus
3.2.2.6
W181F
site-directed mutagenesis, the mutant shows a decrease in activity and an increase in ADP cyclization compared to the wild-type enzyme
Bos taurus
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
3.2.2.6
1,N6-etheno NAD+
-
Bos taurus
3.2.2.6
2'-deoxy-2'-beta-D-fluoroarabinofuranoside NAD+
-
Bos taurus
3.2.2.6
2'-deoxy-2'-beta-D-fluororibofuranoside NAD+
non-covalent complex of the inhibitor formed with enzyme mutant E218Q, PDB ID: 3ghh, and with wild-type enzyme, PDB ID: 3kou
Bos taurus
3.2.2.6
3-aminopyridine
-
Bos taurus
3.2.2.6
Isonicotinic acid hydrazide
-
Bos taurus
3.2.2.6
nicotinamide
inhibition involves enzyme residue Trp181
Bos taurus
3.2.2.6
Pyridine
non-competitive inhibition of mutant E218A by pyridine
Bos taurus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.0171
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0175
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0189
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0215
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0247
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0276
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.028
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0289
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0295
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0297
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.036
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.0453
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.2.2.6
NAD+ + H2O
Bos taurus
bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism
ADP-D-ribose + nicotinamide
-
-
?
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.2.2.6
additional information
selectivity in favor of methanolysis by wild-type enzyme and mutant E138A. 1'-Azido ADP-ribose is the reaction product obtained in the presence of azide. the ADP-ribosyl cyclase activity of wild-type bCD38 isminimal
731357
Bos taurus
?
-
-
-
-
3.2.2.6
NAD+ + H2O
bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism
731357
Bos taurus
ADP-D-ribose + nicotinamide
-
-
-
?
3.2.2.6
NAD+ + H2O
via a stepwise reaction mechanism
731357
Bos taurus
ADP-D-ribose + nicotinamide
-
-
-
?
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.2.2.6
37
-
assay at
Bos taurus
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.0155
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.016
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.03
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.122
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.49
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.81
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
3.5
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
6.64
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
7.8
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
10.36
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
18.37
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
57.9
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.2.2.6
7.4
-
assay at
Bos taurus
General Information
EC Number
General Information
Commentary
Organism
3.2.2.6
additional information
structure-function analysis, overview. The enzyme catalyzes the formation of beta-1'-O-methyl ADP-ribose in presence of methanol, solvolysis does not affect the overall turnover rate of NAD+ by the wild-type enzyme. Precise role of key conserved active site residues Trp118, Glu138, Asp147, Trp181 and Glu218, effects of experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. Binding of 2'-fluorinated analogs of NAD+ and trappping of the reaction intermediate, detailed overview. Catalytic residue Glu138 is part of the TLEDTL signature domain, Asp147 is a highly conserved residue in the enzyme and is important for the catalytic parameters. Cooperative contribution of Trp118 and Trp181 to catalysis
Bos taurus
General Information (protein specific)
EC Number
General Information
Commentary
Organism
3.2.2.6
additional information
structure-function analysis, overview. The enzyme catalyzes the formation of beta-1'-O-methyl ADP-ribose in presence of methanol, solvolysis does not affect the overall turnover rate of NAD+ by the wild-type enzyme. Precise role of key conserved active site residues Trp118, Glu138, Asp147, Trp181 and Glu218, effects of experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. Binding of 2'-fluorinated analogs of NAD+ and trappping of the reaction intermediate, detailed overview. Catalytic residue Glu138 is part of the TLEDTL signature domain, Asp147 is a highly conserved residue in the enzyme and is important for the catalytic parameters. Cooperative contribution of Trp118 and Trp181 to catalysis
Bos taurus
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.54
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.55
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
1.21
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
6.4
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
10.8
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
29.3
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
200
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
220
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
290
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
360
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
630
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
3380
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
3.2.2.6
0.54
-
NAD+
recombinant mutant W118A/W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
0.55
-
NAD+
recombinant mutant E218A, pH 7.4, 37°C
Bos taurus
3.2.2.6
1.21
-
NAD+
recombinant mutant E218Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
6.4
-
NAD+
recombinant mutant W118A, pH 7.4, 37°C
Bos taurus
3.2.2.6
10.8
-
NAD+
recombinant mutant W181A, pH 7.4, 37°C
Bos taurus
3.2.2.6
29.3
-
NAD+
recombinant mutant E138A, pH 7.4, 37°C
Bos taurus
3.2.2.6
200
-
NAD+
recombinant mutant D147A, pH 7.4, 37°C
Bos taurus
3.2.2.6
220
-
NAD+
recombinant mutant E138Q, pH 7.4, 37°C
Bos taurus
3.2.2.6
290
-
NAD+
recombinant mutant W118F, pH 7.4, 37°C
Bos taurus
3.2.2.6
360
-
NAD+
recombinant mutant W181F, pH 7.4, 37°C
Bos taurus
3.2.2.6
630
-
NAD+
recombinant mutant W118H, pH 7.4, 37°C
Bos taurus
3.2.2.6
3380
-
NAD+
recombinant wild-type enzyme, pH 7.4, 37°C
Bos taurus