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Literature summary for 3.2.2.6 extracted from

  • Kuhn, I.; Kellenberger, E.; Cakir-Kiefer, C.; Muller-Steffner, H.; Schuber, F.
    Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues (2014), Biochim. Biophys. Acta, 1844, 1317-1331.
    View publication on PubMed
Search for more literature for 3.2.2.6

Protein Variants

Protein Variants Comment Organism
D147A site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
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
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
E218A site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
E218Q site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
W118A site-directed mutagenesis, the mutant shows a decrease of the catalytic rate compared to the wild-type enzyme Bos taurus
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
W118F site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
W118H site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
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
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

Inhibitors Comment Organism Structure
1,N6-etheno NAD+
-
 Bos taurus
2'-deoxy-2'-beta-D-fluoroarabinofuranoside NAD+
-
 Bos taurus
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-aminopyridine
-
 Bos taurus
Isonicotinic acid hydrazide
-
 Bos taurus
nicotinamide inhibition involves enzyme residue Trp181 Bos taurus
Pyridine non-competitive inhibition of mutant E218A by pyridine Bos taurus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0171
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus
0.0175
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
0.0189
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
0.0215
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
0.0247
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
0.0276
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
0.028
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
0.0289
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
0.0295
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
0.0297
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
0.036
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
0.0453
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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

Organism UniProt Comment Textmining
Bos taurus Q9TTF5
-
-

Reaction

Reaction Comment Organism Reaction ID
NAD+ + H2O = ADP-D-ribose + nicotinamide + H+ 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
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 Bos taurus ?
-
 ?
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 Bos taurus ADP-D-ribose + nicotinamide
-
 ?
NAD+ + H2O via a stepwise reaction mechanism Bos taurus ADP-D-ribose + nicotinamide
-
 ?

Synonyms

Synonyms Comment Organism
bCD38
-
 Bos taurus
CD38/NAD+ glycohydrolase
-
 Bos taurus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Bos taurus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0155
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
0.016
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
0.03
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
0.122
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
0.49
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus
0.81
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
3.5
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
6.64
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
7.8
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
10.36
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
18.37
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
57.9
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
 assay at Bos taurus

General Information

General Information Comment Organism
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]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.54
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
0.55
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
1.21
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
6.4
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
10.8
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus
29.3
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
200
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
220
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
290
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
360
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
630
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
3380
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus