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Literature summary for 1.1.1.67 extracted from

  • Klimacek, M.; Nidetzky, B.
    The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites (2010), Biochem. J., 425, 455-463.
    View publication on PubMed
Search for more literature for 1.1.1.67

Protein Variants

Protein Variants Comment Organism
N191A the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex Pseudomonas fluorescens
N191A/N300A the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, with additive effects in the double mutant Pseudomonas fluorescens
N191L the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex Pseudomonas fluorescens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0033
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
0.017
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
0.023
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
0.055
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
0.067
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens
0.093
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
0.24
-
 D-fructose wild-type, pH 7.1, 25°C Pseudomonas fluorescens
0.31
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
0.314
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
0.4
-
 D-mannitol wild-type, pH 10.0, 25°C Pseudomonas fluorescens
0.9
-
 D-mannitol mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
1.1
-
 D-fructose mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
8.7
-
 D-mannitol mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
9
-
 D-fructose mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
20
-
 D-fructose mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
1187
-
 D-mannitol mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens

Organism

Organism UniProt Comment Textmining
Pseudomonas fluorescens O08355
-
-

Reaction

Reaction Comment Organism Reaction ID
D-mannitol + NAD+ = D-fructose + NADH + H+ the oxyanion hole of mannitol 2-dehydrogenase drives a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is activated for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 in mannitol oxidation and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 in fructose reduction. In the subsequent hydride transfer step, the two asparagine residues provide about 40 kJ/mol of electrostatic stabilization Pseudomonas fluorescens
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-fructose + NADH + H+
-
 Pseudomonas fluorescens D-mannitol + NAD+
-
 ?
D-mannitol + NAD+
-
 Pseudomonas fluorescens D-fructose + NADH + H+
-
 ?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.00045
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
0.04
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
0.55
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
0.55
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
0.56
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
2.78
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
40
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
61
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.000026
-
 D-fructose mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
0.000034
-
 D-mannitol mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
0.019
-
 NADH mutant N191A/N300A, pH 7.1, 25°C Pseudomonas fluorescens
0.064
-
 D-fructose mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
0.127
-
 NAD+ mutant N191A/N300A, pH 10.0, 25°C Pseudomonas fluorescens
0.319
-
 D-mannitol mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
0.407
-
 D-fructose mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
0.598
-
 D-mannitol mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
8.968
-
 NAD+ mutant N191A, pH 10.0, 25°C Pseudomonas fluorescens
9.964
-
 NAD+ mutant N191L, pH 10.0, 25°C Pseudomonas fluorescens
32
-
 NADH mutant N191A, pH 7.1, 25°C Pseudomonas fluorescens
100
-
 D-mannitol wild-type, pH 10.0, 25°C Pseudomonas fluorescens
170
-
 NADH mutant N191L, pH 7.1, 25°C Pseudomonas fluorescens
250
-
 D-fructose wild-type, pH 7.1, 25°C Pseudomonas fluorescens
400
-
 NAD+ wild-type, pH 10.0, 25°C Pseudomonas fluorescens
910
-
 NADH wild-type, pH 7.1, 25°C Pseudomonas fluorescens