Enzyme

3.1.3.1 - Alkaline phosphatase

Alternative Name(s)
  • Glycerophosphatase.
  • Alkaline phosphomonoesterase.
  • Phosphomonoesterase.

Catalytic Activity

phosphate monoester + H2O = an alcohol + phosphate

Cofactors

Mg(2+); Zn(2+).

Reaction Mechanism

    alkaline phosphatase By Reference

    Alkaline phosphatases are zinc and magnesium containing metalloenzymes which function optimally at high pH. They are found in all organisms except some plants, in the periplasmic space (E. coli), in vacuoles (fungi) or GPI-anchored to the external cell membrane (mammals). Alkaline phosphatase-type activity is involved in many cell processes, including metabolism of glycerolipids, folate, and xenobiotics. They cleave phosphomonoester bonds from various substrates, such as the end of a DNA molecule.

    A metal-activated water molecule deprotonates the nucleophilic serine residue. Serine attacks the phosphomonoester in a substitution reaction which eliminates the alcohol. The alcohol anion deprotonates water, which initiates a nucleophilic attack on the covalently bound phosphorus in a substitution reaction which eliminates serine as an anion. Serine then deprotonates water to regenerate the starting state of the enzyme.

    There have been suggestions that the magnesium ion in the active site is essential for catalysis (PMID:10873454), but more recent evidence suggests it is not directly involved in the catalytic mechanism (PMID:18851975).
    Catalytic Residues
    AA Uniprot Uniprot Resid PDB PDB Resid
    Arg P00634 188 1alk 166
    Thr P00634 177 1alk 155
    Glu P00634 344 1alk 322
    Asp P00634 349 1alk 327
    His P00634 353 1alk 331
    Asp P00634 391 1alk 369
    His P00634 392 1alk 370
    His P00634 434 1alk 412
    Asp P00634 175 1alk 153
    Ser P00634 124 1alk 102
    Lys P00634 350 1alk 328
    Asp P00634 73 1alk 51
    Step Components

    dephosphorylation, intermediate formation, intermediate terminated, enzyme-substrate complex formation, proton transfer, native state of enzyme regenerated, enzyme-substrate complex cleavage, overall reactant used, hydrolysis, bimolecular nucleophilic substitution, overall product formed

    Step 1.

    Metal activated water deprotonates Ser102.

    Step 2.

    Ser102 initiates a nucleophilic attack on the phosphoric monoester in a substitution reaction which eliminates the alcohol as an anionic species.

    Step 3.

    The alcohol anion deprotonates water, which initiates a nucleophilic attack on the covalently bound phosphorous in a substitution reaction which eliminates serine as an anion.

    Step 4.

    Ser102 deprotonates water to regenerate the starting state of the enzyme.

    Products.

    The products of the reaction.

Reaction Parameters

  • Kinetic Parameters
    Organism KM Value [mM] Substrate Comment
    Escherichia coli 0.00002 4-nitrophenyl phosphate mutant enzyme D101A/R166S/E322Y/K328A, at pH 8.0 and 25°C
    Burkholderia cenocepacia 40.8 O-phosphoserine
    Vibrio splendidus 67 4-nitrophenyl phosphate with 0.5 M NaCl, at pH 10.5 and 25°C
    Cobetia marina 1200 4-nitrophenyl phosphate 25°C, pH 10.3, Tris-HCl buffer
    Dioszegia sp. 2400 4-nitrophenyl phosphate at pH 9.0 and 47°C
  • Temperature
    Organism Temperature Range Comment
    antarctic bacterium 0 - 25 0°C: 38% of maximal activity, 25°C: optimum
    Dioszegia sp. 4 - 55 4°C: 43% of maximal activity, 10°C: 67% of maximal activity, 30°C: about 88% of maximal activity, 55°C: about 60% of maximal activity
    Bacillus sp. (in: Bacteria) 5 - 60 5°C: 27% of maximal activity, 60°C: about 60% of maximal activity
    Scrobicularia plana 20 - 60
    Penicillium chrysogenum 20 - 60 20°C: about 35% of maximal activity, 60°C: about 30% of maximal activity
  • pH
    Organism pH Range Comment
    Escherichia coli 5 - 10 activity profile of free enzyme is comparable to immobilized enzyme
    Homo sapiens 6 - 9 pH 6.0: about 30% of maximal activity, pH 9.0: about 15% of maximal activity
    Penaeus merguiensis 6 - 12 the enzyme retains a high activity (more than 50%) in the pH range from pH 6.0-12.0
    Cyberlindnera jadinii 7 - 10 pH 7.0: about 20% of maximal activity, pH 10.0: about 90% of maximal activity
    Geobacillus stearothermophilus 7 - 10.5 pH 7.0: about 10% of maximal activity, pH 10.5: about 35% of maximal activity

Associated Proteins

Protein name Organism
Alkaline phosphatase L Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Alkaline phosphatase 4 Fruit fly
Alkaline phosphatase PhoV Synechococcus elongatus (strain PCC 7942 / FACHB-805)
Alkaline phosphatase, germ cell type Human
Alkaline phosphatase, tissue-nonspecific isozyme Human

Citations