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Literature summary for 3.1.3.1 extracted from

  • Zalatan, J.G.; Fenn, T.D.; Herschlag, D.
    Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal ion (2008), J. Mol. Biol., 384, 1174-1189.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.3.1

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli as an N-terminal maltose binding protein (MBP) fusion construct (AP-MBP) Escherichia coli

Protein Variants

Protein Variants Comment Organism
E22Y kcat/KM (1/M*sec) (4-nitrophenyl phosphate): 7200, (3-nitrobenzyl phosphate): 31, (methyl phosphate): 1.6, (methyl 4-nitrophenyl phosphate): 35, (bis-4-nitrophenyl phosphate): 0.07, (4-nitrophenyl sulfate): 0.0000029 Escherichia coli
R166S kcat/KM (1/M*sec) (4-nitrophenyl phosphate): 100000, (3-nitrobenzyl phosphate): 2300, (methyl phosphate): 110, (methyl 4-nitrophenyl phosphate): 0.48, (bis-4-nitrophenyl phosphate): 0.05, (4-nitrophenyl sulfate): 0.000058 Escherichia coli
R166S/E322Y kcat/KM (1/M*sec) (4-nitrophenyl phosphate): 1.6, (3-nitrobenzyl phosphate): below 0.2, (methyl phosphate): not determined, (methyl 4-nitrophenyl phosphate): 0.24, (bis-4-nitrophenyl phosphate): 0.021, (4-nitrophenyl sulfate): below 0.000001 Escherichia coli
W322A kcat/KM (1/M*sec) (4-nitrophenyl phosphate): 8900, (3-nitrobenzyl phosphate): not determined, (methyl phosphate): not determined, (methyl 4-nitrophenyl phosphate): 18, (bis-4-nitrophenyl phosphate): 0.037, (4-nitrophenyl sulfate): not determined Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ removal of a third metal ion site near the bimetallo site, containing Mg2+, suggests that the Mg2+ ion participates in general base catalysis. Mg2+ ion stabilizes the transferred phosphoryl group in the transition state, and this interaction is distinct from those mediated by the Zn2+ bimetallo site. Positioning of charged or polar groups to interact with all three nonbridging oxygen atoms of the transferred phosphoryl group is important for catalysis of phosphate monoester hydrolysis Escherichia coli
Zn2+ removal of a third metal ion site near the bimetallo site, containing Mg2+, suggests that the Mg2+ ion participates in general base catalysis. Mg2+ ion stabilizes the transferred phosphoryl group in the transition state, and this interaction is distinct from those mediated by the Zn2+ bimetallo site. Positioning of charged or polar groups to interact with all three nonbridging oxygen atoms of the transferred phosphoryl group is important for catalysis of phosphate monoester hydrolysis Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P00634
-
-

Purification (Commentary)

Purification (Comment) Organism
using affinity chromatography. Typical yields for a 6-L culture are 30-40 mg of pure protein Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-nitrobenzyl phosphate + H2O
-
 Escherichia coli 3-nitrobenzoate + phosphate
-
 ?
4-nitrophenyl phosphate + H2O
-
 Escherichia coli 4-nitrophenol + phosphate
-
 ?
4-nitrophenyl sulfate + H2O
-
 Escherichia coli 4-nitrophenol + sulfate
-
 ?
bis-4-nitrophenyl phosphate + H2O
-
 Escherichia coli 4-nitrophenol + phosphate
-
 ?
methyl 4-nitrophenyl phosphate + H2O
-
 Escherichia coli methyl phosphate + 4-nitrophenol
-
 ?
methyl 4-nitrophenyl phosphorothioate + H2O only the R-enantiomer is detectably hydrolyzed by the enzyme Escherichia coli 4-nitrophenol + methyl phosphorothioate
-
 ?
methyl phosphate + H2O
-
 Escherichia coli methanol + 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 wild-type enzyme: kcat/KM (1/M*sec) (4-nitrophenyl phosphate): 33000000, (3-nitrobenzyl phosphate): 18000000, (methyl phosphate): 1200000, (methyl 4-nitrophenyl phosphate): 18, (bis-4-nitrophenyl phosphate): 0.05, (4-nitrophenyl sulfate): 0.01 Escherichia coli

pH Optimum

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