Adenine phosphoribosyltransferase

 

Adenine phosphoribosyltransferase (APRTase) catalyses the reversible Mg2+ dependent reaction of adenine with 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce AMP and pyrophosphate. This reaction is important in adenine salvage and recycling, and the enzyme is present in species ranging from bacteria to mammals. In humans, APRTase has the sole metabolic function of recycling adenine formed in the polyamine pathway, and the effects of APRTase deficiency are relatively mild. Some protozoan parasite, including Giardia lamblia, are deficient in de novo purine synthesis and so purine uptake from the host and the APRTase reaction are especially important in these organisms.

 

Reference Protein and Structure

Sequence
Q967M2 UniProt IPR000836 (Sequence Homologues) (PDB Homologues)
Biological species
Giardia intestinalis (Flagellated parasite) Uniprot
PDB
1l1r - Crystal Structure of APRTase from Giardia lamblia Complexed with 9-deazaadenine, Mg2+ and PRPP (1.95 Å) PDBe PDBsum 1l1r
Catalytic CATH Domains
3.40.50.2020 CATHdb (see all for 1l1r)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.7)

5-O-phosphonato-alpha-D-ribofuranosyl diphosphate(5-)
CHEBI:58017ChEBI
+
adenine
CHEBI:16708ChEBI
→
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: AMP pyrophosphorylase, AMP-pyrophosphate phosphoribosyltransferase, APRT, Adenine phosphoribosylpyrophosphate transferase, Adenosine phosphoribosyltransferase, Adenylate pyrophosphorylase, Adenylic pyrophosphorylase, Transphosphoribosidase, AMP diphosphorylase,

Enzyme Mechanism

Introduction

In the proposed Sn1 mechanism, Glu100 functions as a general base to deprotonate N7 of adenine. This enables the N7 of adenine to nucleophilically attack the electrophilic carbon. The reaction passes through an oxacarbenium-like transition state in which the Mg2+ ion and Arg 63 stabilise accumulation of negative charge on the pyrophosphate leaving group.

Catalytic Residues Roles

UniProt PDB* (1l1r)
Arg63 Arg63A Stabilises accumulation of negative charge on the pyrophosphate leaving group. electrostatic stabiliser
Glu100 Glu100A Deprotonates N7 of adenine. proton acceptor, proton donor
*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, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Shi W et al. (2002), J Biol Chem, 277, 39981-39988. Closed Site Complexes of Adenine Phosphoribosyltransferase fromGiardia lamblia Reveal a Mechanism of Ribosyl Migration. DOI:10.1074/jbc.m205596200. PMID:12171925.
  2. Arco JD et al. (2017), Curr Pharm Des,Purine and Pyrimidine Phosphoribosytransferases: A versatile tool for enzymatic synthesis of nucleoside-5'-monophosphates. DOI:10.2174/1381612823666171017165707. PMID:29046144.
  3. Takahashi R et al. (2010), J Biochem, 147, 95-107. Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase. DOI:10.1093/jb/mvp152. PMID:19819904.
  4. Shi W et al. (2001), Biochemistry, 40, 10800-10809. Structural Analysis of Adenine Phosphoribosyltransferase fromSaccharomyces cerevisiae†,‡. DOI:10.1021/bi010465h. PMID:11535055.

Step 1. Glu100 deprotonates N7 of adenine, increasing the base's nucleophilicity to attack the anomeric carbon on the ribose sugar in an SN1 like reaction with an oxocarbeniom ion like transition state which is stabilised by Arg63.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg63A electrostatic stabiliser
Glu100A proton acceptor

Chemical Components

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

Step 2. Glu100 is deprotonated by the pyrophosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu100A proton donor

Chemical Components

overall product formed, proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu100 deprotonates N7 of adenine, increasing the base's nucleophilicity to attack the anomeric carbon on the ribose sugar in an SN1 like reaction with an oxocarbeniom ion like transition state which is stabilised by Arg63.

Step 2. Glu100 is deprotonated by the pyrophosphate product.

Products.

Introduction

In the proposed Sn1 reaction, there is bond cleavage between the ribose and the pyrophosphate to form an oxocarbenium ion intermediate. This intermediate is subsequently attacked by N7 nucleophile on the adenine. Arg63 still stabilises the negatively charged pyrophosphate leaving group.

Catalytic Residues Roles

UniProt PDB* (1l1r)
Arg63 Arg63A Stabilises accumulation of negative charge on the pyrophosphate leaving group.
Glu100 Glu100A Deprotonates N7 of adenine. proton acceptor, proton donor
*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

elimination (not covered by the Ingold mechanisms), heterolysis, intermediate formation, bimolecular nucleophilic addition, proton transfer, overall reactant used, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Arco JD et al. (2017), Curr Pharm Des,Purine and Pyrimidine Phosphoribosytransferases: A versatile tool for enzymatic synthesis of nucleoside-5'-monophosphates. DOI:10.2174/1381612823666171017165707. PMID:29046144.
  2. Takahashi R et al. (2010), J Biochem, 147, 95-107. Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase. DOI:10.1093/jb/mvp152. PMID:19819904.
  3. Silva CH et al. (2008), J Biomol Struct Dyn, 25, 589-597. Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism. DOI:10.1080/07391102.2008.10507205. PMID:18399692.
  4. Shi W et al. (2002), J Biol Chem, 277, 39981-39988. Closed Site Complexes of Adenine Phosphoribosyltransferase fromGiardia lamblia Reveal a Mechanism of Ribosyl Migration. DOI:10.1074/jbc.m205596200. PMID:12171925.
  5. Shi W et al. (2001), Biochemistry, 40, 10800-10809. Structural Analysis of Adenine Phosphoribosyltransferase fromSaccharomyces cerevisiae†,‡. DOI:10.1021/bi010465h. PMID:11535055.

Step 1. Leaving group departure before nucleobase approaches.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

elimination (not covered by the Ingold mechanisms), heterolysis, intermediate formation

Step 2. Glu100 activates N7 by deprotonation to behave as a nucleophile and attack the oxocarbenium ion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu100A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used

Step 3. Pyrophosphate deprotonates Glu100 to regenerate active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu100A proton donor

Chemical Components

overall product formed, inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Leaving group departure before nucleobase approaches.

Step 2. Glu100 activates N7 by deprotonation to behave as a nucleophile and attack the oxocarbenium ion intermediate.

Step 3. Pyrophosphate deprotonates Glu100 to regenerate active site.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Morwenna Hall