Purple acid phosphatase

 

Purple acid phosphatases (PAPs) are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters (e.g. of sugars or proteins) and anhydrides (e.g. ATP) under acidic conditions. The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II/III) in mammals or a Zn(II) or Mn(II) in plants.

Crystal structures of human, pig, rat, and plant PAPs have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.

 

Reference Protein and Structure

Sequence
P80366 UniProt (3.1.3.2) IPR015914 (Sequence Homologues) (PDB Homologues)
Biological species
Phaseolus vulgaris (Kidney bean) Uniprot
PDB
4kbp - KIDNEY BEAN PURPLE ACID PHOSPHATASE (2.7 Å) PDBe PDBsum 4kbp
Catalytic CATH Domains
3.60.21.10 CATHdb (see all for 4kbp)
Cofactors
Iron(3+) (1), Zinc(2+) (1), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.3.2)

water
CHEBI:15377ChEBI
+
phosphate monoester dianion
CHEBI:67140ChEBI
→
alcohol
CHEBI:30879ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: Acid monophosphatase, Acid nucleoside diphosphate phosphatase, Acid phosphohydrolase, Acid phosphomonoester hydrolase, Acid phosphomonoesterase, Glycerophosphatase, Orthophosphoric-monoester phosphohydrolase (acid optimum), Phosphomonoesterase, Uteroferrin,

Enzyme Mechanism

Introduction

On the basis of structural studies with bound phosphate and tungstate (inhibitor), and the observed inversion of the phosphorous configuration during catalysis, an SN2-type mechanism is proposed for PAP. The substrate phosphate is bound to the Me(II) ion via one of the non-esterified oxygen atoms and oriented by His202 and His296 (numbering for kidney bean enzyme) for in-line attack of an Fe(III)-bound hydroxide ion from a position opposite the esterified oxygen. These residues and the metal ions are presumed to stabilise the pentaco-ordinate transition state. Tentatively, His296 may then act as a general acid to protonate the leaving group, followed by the attack of a water molecule on the Fe(III) atom to release the inorganic phosphate.

Catalytic Residues Roles

UniProt PDB* (4kbp)
His322, His229 His295A, His202A Help stabilise the negatively charged intermediates and transition states. hydrogen bond donor, electrostatic stabiliser
His323 His296A General acid/base. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
His352, Asp191, Asp162, Tyr194 His325A, Asp164A, Asp135A, Tyr167A Form the iron binding site. metal ligand
Asp191, Asn228, His313, His350 Asp164A, Asn201A, His286A, His323A Form the zinc 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

overall reactant used, intermediate formation, proton transfer, bimolecular nucleophilic substitution, coordination to a metal ion, decoordination from a metal ion, native state of enzyme regenerated

References

  1. Klabunde T et al. (1996), J Mol Biol, 259, 737-748. Mechanism of Fe(III) – Zn(II) Purple Acid Phosphatase Based on Crystal Structures. DOI:10.1006/jmbi.1996.0354. PMID:8683579.
  2. Feder D et al. (2012), Chem Biol Drug Des, 80, 665-674. Identification of Purple Acid Phosphatase Inhibitors by Fragment-Based Screening: Promising New Leads for Osteoporosis Therapeutics. DOI:10.1111/cbdd.12001. PMID:22943065.
  3. Anand A et al. (2012), Appl Biochem Biotechnol, 167, 2174-2197. A Molecular Description of Acid Phosphatase. DOI:10.1007/s12010-012-9694-8. PMID:22684363.
  4. Schenk G et al. (2012), Acc Chem Res, 45, 1593-1603. Binuclear Metallohydrolases: Complex Mechanistic Strategies for a Simple Chemical Reaction. DOI:10.1021/ar300067g. PMID:22698580.
  5. Schenk G et al. (2005), Proc Natl Acad Sci U S A, 102, 273-278. Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase. DOI:10.1073/pnas.0407239102. PMID:15625111.
  6. Sträter N et al. (1995), Science, 268, 1489-1492. Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site. PMID:7770774.

Step 1. Metal activated water attacks the phosphate in a nucleophilic addition, resulting in a pentavalent transition state, which collapses to release the alcohol group, which gains a proton from His296.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His202A hydrogen bond donor, electrostatic stabiliser
His296A hydrogen bond donor, electrostatic stabiliser
Asp135A metal ligand
Asp164A metal ligand
Tyr167A metal ligand
His325A metal ligand
Asn201A metal ligand
His286A metal ligand
His323A metal ligand
His296A proton donor

Chemical Components

overall reactant used, intermediate formation, proton transfer, ingold: bimolecular nucleophilic substitution

Step 2. The phosphate undergoes a rotation in the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His295A hydrogen bond donor, electrostatic stabiliser
His202A hydrogen bond donor, electrostatic stabiliser
His296A hydrogen bond donor
His296A electrostatic stabiliser
Asp135A metal ligand
Asp164A metal ligand
Tyr167A metal ligand
His325A metal ligand
Asn201A metal ligand
His286A metal ligand
His323A metal ligand

Chemical Components

Step 3. Water displaces one of the phosphate coordination bonds.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His202A hydrogen bond donor, electrostatic stabiliser
His295A hydrogen bond donor, electrostatic stabiliser
His296A hydrogen bond acceptor
Asp135A metal ligand
Asp164A metal ligand
Tyr167A metal ligand
His325A metal ligand
Asn201A metal ligand
His286A metal ligand
His323A metal ligand
His296A electrostatic stabiliser

Chemical Components

coordination to a metal ion, decoordination from a metal ion

Step 4. The metal coordinated water is now deprotonated by the leaving phosphate group, regenerating the active site. In the product release step, another water enters the active site, displacing the phosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp135A metal ligand
Asp164A metal ligand
Tyr167A metal ligand
His325A metal ligand
Asn201A metal ligand
His286A metal ligand
His323A metal ligand
His296A electrostatic stabiliser
His295A electrostatic stabiliser
His202A electrostatic stabiliser

Chemical Components

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

Step 1. Metal activated water attacks the phosphate in a nucleophilic addition, resulting in a pentavalent transition state, which collapses to release the alcohol group, which gains a proton from His296.

Step 2. The phosphate undergoes a rotation in the active site.

Step 3. Water displaces one of the phosphate coordination bonds.

Step 4. The metal coordinated water is now deprotonated by the leaving phosphate group, regenerating the active site. In the product release step, another water enters the active site, displacing the phosphate product.

Products.

Contributors

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