Purine-nucleoside phosphorylase

 

Purine-nucleoside phosphorylase (PNP) catalyses the reversible phosphorolysis of purine nucleosides to generate the corresponding purine base and ribose 1-phosphate. There are two types of this enzyme, showing reasonably high sequence similarity within families and little identity between them. Type I (of which this entry is an example) tend to be trimeric and are found mainly in mammals. Type I enzymes are specific for inosine/guanine nucleosides and have a molecular mass of around 90kDa. Type II tend to be hexameric (although some are thought to be tetramers) and have broad substrate specificity. Type II enzymes are found mainly in prokaryotes and have a molecular mass between 110 and 150 kDa. There are also some PNP enzymes which fall into neither class.

 

Reference Protein and Structure

Sequence
P00491 UniProt (2.4.2.1) IPR011270 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1rr6 - Structure of human purine nucleoside phosphorylase in complex with Immucillin-H and phosphate (2.5 Å) PDBe PDBsum 1rr6
Catalytic CATH Domains
3.40.50.1580 CATHdb (see all for 1rr6)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.1)

inosine
CHEBI:17596ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
→
alpha-D-ribose 1-phosphate(2-)
CHEBI:57720ChEBI
+
hypoxanthine
CHEBI:17368ChEBI
Alternative enzyme names: PNPase, PUNPI, PUNPII, Inosine phosphorylase, Inosine-guanosine phosphorylase, Nucleotide phosphatase, Purine deoxynucleoside phosphorylase, Purine deoxyribonucleoside phosphorylase, Purine nucleoside phosphorylase, Purine ribonucleoside phosphorylase,

Enzyme Mechanism

Introduction

The phosphate group is di-anionic and is stabilised by the Ser33-His64-His86 catalytic triad. It attacks the electrophilic C1 involved in the glycosidic bond in an SN2 manner. His257, Tyr88, Met119 all stabilise the ribose ring transition state while Ala116 and Ser220 also stabilise alongside the triad the phosphate transition state. Hypoxanthine is then protonated by the phosphoryl proton via the 2' and 3' OH groups on the ribose, although there are multiple pathways that protonation can take place through.

Catalytic Residues Roles

UniProt PDB* (1rr6)
Ser220 Ser220A Stabilise phosphate by a side chain hydrogen bond. hydrogen bond donor, electrostatic stabiliser
Ala116 (main-N), Ser33 (main-N) Ala116A (main-N), Ser33A (main-N) Stabilise and position the phosphate group through their main chain amide groups. hydrogen bond donor, electrostatic stabiliser
Tyr88, His257, Met219 (main-N) Tyr88A, His257A, Met219A (main-N) Stabilise the ribose ring in the transition state via hydrogen bonds. hydrogen bond donor, electrostatic stabiliser
His64, Ser33, His86 His64A, Ser33A, His86A Catalytic triad stabilises the nucleophilic phosphate. electrostatic stabiliser
Asn243 Asn243A Stabilises base via hydrogen bonding, thought to also be involved in determining substrate specificity. electrostatic stabiliser, polar interaction
*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

bimolecular nucleophilic substitution, rate-determining step, heterolysis, overall reactant used, proton relay, overall product formed, proton transfer

References

  1. Isaksen GV et al. (2016), Biochemistry, 55, 2153-2162. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase. DOI:10.1021/acs.biochem.5b01347. PMID:26985580.
  2. Shi W et al. (2004), J Biol Chem, 279, 18103-18106. Plasmodium falciparum purine nucleoside phosphorylase: crystal structures, immucillin inhibitors, and dual catalytic function. DOI:10.1074/jbc.C400068200. PMID:14982926.

Step 1. The incoming nucleophilic phosphate group is stabilised by the Ser33-His64-His86 catalytic triad. O4 on the phosphate attacks the glycosidic C1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His86A hydrogen bond acceptor
Ser33A electrostatic stabiliser
His64A electrostatic stabiliser
Asn243A electrostatic stabiliser
Met219A (main-N) electrostatic stabiliser
Ser220A electrostatic stabiliser, hydrogen bond donor
Tyr88A electrostatic stabiliser
His257A electrostatic stabiliser, hydrogen bond acceptor
Tyr88A hydrogen bond donor
Met219A (main-N) hydrogen bond donor
Ala116A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser33A hydrogen bond donor
Ser33A (main-N) hydrogen bond donor
Asn243A polar interaction

Chemical Components

ingold: bimolecular nucleophilic substitution, rate-determining step, heterolysis, overall reactant used

Step 2. Proton shuttle from the phosphate group via the 2' and 3' OH on the ribose sugar to N9 on hypoxanthine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

proton relay, overall product formed, proton transfer
Download: Image, Marvin File

Step 1. The incoming nucleophilic phosphate group is stabilised by the Ser33-His64-His86 catalytic triad. O4 on the phosphate attacks the glycosidic C1.

Step 2. Proton shuttle from the phosphate group via the 2' and 3' OH on the ribose sugar to N9 on hypoxanthine.

Products.

Introduction

Proposed mechanism in which the phosphate is activated by His86 and attacks the ribose ring directly via an SN2 type reaction.

Catalytic Residues Roles

UniProt PDB* (1rr6)
His86 His86A Acts as a general acid/base, abstracting a proton from the phosphate substrate and being returned to its initial protonation state by the purine product. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu89 Glu89A Activates the catalytic His. activator, hydrogen bond acceptor, electrostatic stabiliser
Asn243 Asn243A Stabilises the negatively charged intermediate. hydrogen bond donor, electrostatic stabiliser
*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, overall reactant used, intermediate formation, heterolysis, charge delocalisation, intermediate collapse, overall product formed, native state of enzyme regenerated, intermediate terminated

References

  1. Canduri F et al. (2005), Biochem Biophys Res Commun, 327, 646-649. New catalytic mechanism for human purine nucleoside phosphorylase. DOI:10.1016/j.bbrc.2004.12.052. PMID:15649395.
  2. Craig SP 3rd et al. (2000), J Biol Chem, 275, 20231-20234. Purine Phosphoribosyltransferases. DOI:10.1074/jbc.r000002200. PMID:10816600.
  3. Mao C et al. (1998), Biochemistry, 37, 7135-7146. Calf Spleen Purine Nucleoside Phosphorylase Complexed with Substrates and Substrate Analogues†,‡. DOI:10.1021/bi9723919. PMID:9585525.
  4. Koellner G et al. (1997), J Mol Biol, 265, 202-216. Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 Å resolution. DOI:10.1006/jmbi.1996.0730. PMID:9020983.

Step 1. His86 deprotonates phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor, activator
His86A hydrogen bond donor, hydrogen bond acceptor
Asn243A hydrogen bond donor
His86A proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The phosphate initiates a nucleophilic attack on the substrate, resulting in C-N bond cleavage.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor, electrostatic stabiliser
His86A hydrogen bond donor
Asn243A electrostatic stabiliser

Chemical Components

heterolysis, overall reactant used, charge delocalisation, intermediate collapse

Step 3. The purine deprotonates His86.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor
His86A hydrogen bond donor
Asn243A electrostatic stabiliser, hydrogen bond donor
His86A proton donor

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated, intermediate terminated
Download: Image, Marvin File

Step 1. His86 deprotonates phosphate.

Step 2. The phosphate initiates a nucleophilic attack on the substrate, resulting in C-N bond cleavage.

Step 3. The purine deprotonates His86.

Products.

Introduction

The substitution reaction occurs by a SN1 type mechanism. His86 is hydrogen bonded to Glu89, and abstracts a proton from the phosphate ion which causes strain and weakening of the glycosidic bond. The glycosidic bond is cleaved, forming an oxycarbenium ion intermediate which is stabilised by the phosphate dianion. Therefore, the phosphate anion is used for both the initiation of bond cleavage and stabilisation of intermediate [PMID:9305963].

Catalytic Residues Roles

UniProt PDB* (1rr6)
His86 His86A Acts as a general acid/base, abstracting a proton from the substrate phosphate. It is returned to its initial protonation state by the purine product. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Glu89 Glu89A Activates the catalytic His. activator, hydrogen bond acceptor, electrostatic stabiliser
Asn243 Asn243A Helps stabilise the negatively charged intermediates. hydrogen bond donor, electrostatic stabiliser
*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, overall reactant used, intermediate formation, heterolysis, charge delocalisation, elimination (not covered by the Ingold mechanisms), bimolecular nucleophilic addition, overall product formed, intermediate terminated, native state of enzyme regenerated

References

  1. Erion MD et al. (1997), Biochemistry, 36, 11735-11748. Purine Nucleoside Phosphorylase. 2. Catalytic Mechanism†. DOI:10.1021/bi961970v. PMID:9305963.
  2. Isaksen GV et al. (2016), Biochemistry, 55, 2153-2162. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase. DOI:10.1021/acs.biochem.5b01347. PMID:26985580.
  3. Canduri F et al. (2005), Biochem Biophys Res Commun, 327, 646-649. New catalytic mechanism for human purine nucleoside phosphorylase. DOI:10.1016/j.bbrc.2004.12.052. PMID:15649395.
  4. Mao C et al. (1998), Biochemistry, 37, 7135-7146. Calf Spleen Purine Nucleoside Phosphorylase Complexed with Substrates and Substrate Analogues†,‡. DOI:10.1021/bi9723919. PMID:9585525.
  5. Koellner G et al. (1998), J Mol Biol, 280, 153-166. Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (sulphate) at 2.1 Å resolution. DOI:10.1006/jmbi.1998.1799. PMID:9653038.
  6. Koellner G et al. (1997), J Mol Biol, 265, 202-216. Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 Å resolution. DOI:10.1006/jmbi.1996.0730. PMID:9020983.
  7. Erion MD et al. (1997), Biochemistry, 36, 11725-11734. Purine Nucleoside Phosphorylase. 1. Structure−Function Studies†. DOI:10.1021/bi961969w. PMID:9305962.

Step 1. His86 deprotonates phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor, activator
His86A hydrogen bond donor, hydrogen bond acceptor
Asn243A hydrogen bond donor
His86A proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. In a heterolysis reaction, the C-N bond between ribose and the purine is cleaved.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor, electrostatic stabiliser
His86A hydrogen bond donor
Asn243A electrostatic stabiliser

Chemical Components

heterolysis, overall reactant used, charge delocalisation, intermediate formation, elimination (not covered by the Ingold mechanisms)

Step 3. Phosphate initiates a nucleophilic attack on the ribose in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor, electrostatic stabiliser
His86A hydrogen bond donor, electrostatic stabiliser
Asn243A electrostatic stabiliser, hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, overall product formed, intermediate terminated

Step 4. The purine deprotonates His86.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu89A hydrogen bond acceptor
His86A hydrogen bond donor
Asn243A electrostatic stabiliser, hydrogen bond donor
His86A proton donor

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated, intermediate terminated
Download: Image, Marvin File

Step 1. His86 deprotonates phosphate.

Step 2. In a heterolysis reaction, the C-N bond between ribose and the purine is cleaved.

Step 3. Phosphate initiates a nucleophilic attack on the ribose in an addition reaction.

Step 4. The purine deprotonates His86.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Sophie T. Williams, Christian Drew, Craig Porter, Katherine Ferris, Morwenna Hall