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Literature summary for 3.1.3.1 extracted from

  • OBrien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.
    Arginine coordination in enzymatic phosphoryl transfer: evaluation of the effect of Arg166 mutations in Escherichia coli alkaline phosphatase (2008), Biochemistry, 47, 7663-7672.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
a 2.1 A X-ray diffraction structure of Arg166Ser alkaline phosphatase is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate Escherichia coli

Protein Variants

Protein Variants Comment Organism
additional information the results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, a free energy profile for the hydrolysis of the enzyme-phosphate intermediate is constructed. This analysis indicates that the arginine side chain strengthens binding by approximately 3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state Escherichia coli
R166A kcat (1/sec) (substrate: 4-nitrophenyl phosphate): 0.3 Escherichia coli
R166K kcat (1/sec) (substrate: 4-nitrophenyl phosphate): 0.65 Escherichia coli
R166S kcat (1/sec) (substrate: 4-nitrophenyl phosphate): 0.5 Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
inorganic phosphate
-
Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
Escherichia coli
Zn2+
-
Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P00634
-
-

Reaction

Reaction Comment Organism Reaction ID
a phosphate monoester + H2O = an alcohol + phosphate Arg166 contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl phosphate + H2O
-
Escherichia coli 4-nitrophenol + phosphate
-
?
ethyl phosphate + H2O
-
Escherichia coli ethanol + phosphate
-
?

Synonyms

Synonyms Comment Organism
alkaline phosphatase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
additional information kcat/KM (1/Msec) wild-type: 140000, mutant R166K: 20, mutant R166S: 24 Escherichia coli
0.3
-
4-nitrophenyl phosphate pH 8.0, 25°C, mutant R166A Escherichia coli
0.5
-
4-nitrophenyl phosphate pH 8.0, 25°C, mutant R166S Escherichia coli
0.65
-
4-nitrophenyl phosphate pH 8.0, 25°C, mutant R166K Escherichia coli
12
-
4-nitrophenyl phosphate pH 8.0, 25°C, wild-type Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.0011
-
inorganic phosphate pH 8.0, 25°C, wild-type Escherichia coli
0.075
-
inorganic phosphate pH 8.0, 25°C, mutant R166K Escherichia coli
0.46
-
inorganic phosphate pH 8.0, 25°C, mutant R166S Escherichia coli
0.65
-
inorganic phosphate pH 8.0, 25°C, mutant R166A Escherichia coli