Alkaline phosphatase

 

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.

 

Reference Protein and Structure

Sequence
P00634 UniProt (3.1.3.1) IPR001952 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1alk - REACTION MECHANISM OF ALKALINE PHOSPHATASE BASED ON CRYSTAL STRUCTURES. TWO METAL ION CATALYSIS (2.0 Å) PDBe PDBsum 1alk
Catalytic CATH Domains
3.40.720.10 CATHdb (see all for 1alk)
Cofactors
Magnesium(2+) (1), Zinc(2+) (2), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.3.1)

water
CHEBI:15377ChEBI
+
phosphate monoester dianion
CHEBI:67140ChEBI
→
alcohol
CHEBI:30879ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: Alkaline phenyl phosphatase, Alkaline phosphohydrolase, Alkaline phosphomonoesterase, Glycerophosphatase, Orthophosphoric-monoester phosphohydrolase (alkaline optimum), Phosphomonoesterase,

Enzyme Mechanism

Introduction

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 Roles

UniProt PDB* (1alk)
Arg188 Arg166A Stabilises transition states. activator, hydrogen bond donor, electrostatic stabiliser
Asp349, His353, His434 Asp327A, His331A, His412A Form the zinc 1 binding site. metal ligand
Ser124 Ser102A Serine is the main nucleophile in the reaction, and forms a covalent intermediate with the substrate. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor, nucleophile, nucleofuge
Thr177, Glu344, Asp175, Lys350, Asp73 Thr155A, Glu322A, Asp153A, Lys328A, Asp51A Form the magnesium binding site. metal ligand
Asp391, His392, Asp73 Asp369A, His370A, Asp51A Form the zinc 2 binding site metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

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

References

  1. Stec B et al. (2000), J Mol Biol, 299, 1303-1311. A revised mechanism for the alkaline phosphatase reaction involving three metal ions. DOI:10.1006/jmbi.2000.3799. PMID:10873454.
  2. Bobyr E et al. (2012), J Mol Biol, 415, 102-117. High-Resolution Analysis of Zn2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography. DOI:10.1016/j.jmb.2011.10.040. PMID:22056344.
  3. Koutsioulis D et al. (2010), Protein Sci, 19, 75-84. Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases. DOI:10.1002/pro.284. PMID:19916164.
  4. Zalatan JG et al. (2008), J Mol Biol, 384, 1174-1189. Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion. DOI:10.1016/j.jmb.2008.09.059. PMID:18851975.
  5. Wang J et al. (2006), Protein Sci, 15, 2395-2401. Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine. DOI:10.1110/ps.062351506. PMID:17008720.
  6. Wang J et al. (2005), Biochemistry, 44, 8378-8386. Metal Specificity Is Correlated with Two Crucial Active Site Residues inEscherichia coliAlkaline Phosphatase†,‡. DOI:10.1021/bi050155p. PMID:15938627.
  7. Holtz KM et al. (1999), J Biol Chem, 274, 8351-8354. A Model of the Transition State in the Alkaline Phosphatase Reaction. DOI:10.1074/jbc.274.13.8351. PMID:10085061.
  8. Stec B et al. (1998), J Mol Biol, 277, 647-662. Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile ser102. DOI:10.1006/jmbi.1998.1635. PMID:9533886.
  9. Murphy JE et al. (1997), Nat Struct Biol, 4, 618-622. Trapping and visualization of a covalent enzyme–phosphate intermediate. DOI:10.1038/nsb0897-618. PMID:9253408.
  10. Ma L et al. (1994), J Biol Chem, 269, 31614-31619. Mutations at histidine 412 alter zinc binding and eliminate transferase activity in Escherichia coli alkaline phosphatase. PMID:7989332.

Step 1. Metal activated water deprotonates Ser102.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser102A hydrogen bond donor
Arg166A activator
Ser102A metal ligand
Asp51A metal ligand
Asp369A metal ligand
His370A metal ligand
Asp327A metal ligand
His412A metal ligand
His331A metal ligand
Thr155A metal ligand
Asp153A metal ligand
Lys328A metal ligand
Glu322A metal ligand
Ser102A proton donor

Chemical Components

proton transfer

Step 2. Ser102 initiates a nucleophilic attack on the phosphoric monoester in a substitution reaction which eliminates the alcohol as an anionic species.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg166A electrostatic stabiliser, hydrogen bond donor
Ser102A metal ligand
Asp51A metal ligand
Asp369A metal ligand
His370A metal ligand
Asp327A metal ligand
His412A metal ligand
His331A metal ligand
Thr155A metal ligand
Asp153A metal ligand
Lys328A metal ligand
Glu322A metal ligand
Ser102A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation

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.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg166A electrostatic stabiliser, hydrogen bond donor
Ser102A metal ligand
Asp51A metal ligand
Asp369A metal ligand
His370A metal ligand
Asp327A metal ligand
His412A metal ligand
His331A metal ligand
Thr155A metal ligand
Asp153A metal ligand
Lys328A metal ligand
Glu322A metal ligand
Ser102A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, overall product formed, enzyme-substrate complex cleavage, dephosphorylation, intermediate terminated, hydrolysis, proton transfer

Step 4. Ser102 deprotonates water to regenerate the starting state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser102A hydrogen bond acceptor
Arg166A electrostatic stabiliser, hydrogen bond donor
Ser102A metal ligand
Asp51A metal ligand
Asp369A metal ligand
His370A metal ligand
Asp327A metal ligand
His412A metal ligand
His331A metal ligand
Thr155A metal ligand
Asp153A metal ligand
Lys328A metal ligand
Glu322A metal ligand
Ser102A proton acceptor

Chemical Components

native state of enzyme regenerated, proton transfer
Download: Image, Marvin File

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.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Stuart Lucas, Craig Porter