Sulfate adenylyltransferase

 

ATP sulphurylase from Saccharomyces cerevisiae is able to catalyse the formation of adenosine-5-phosphosulphate. This functions as the first step in the incorporation of sulphate into biological molecules because it activates the sulphate towards nucleophilic attack by adding a good leaving group. The enzyme is part of a family of ATP sulphurylases (alpha/beta phosphodiesterase superfamily) which include those from the sulphur reducing bacteria, and proteins with dual kinase and sulphurylase activity found in mammals. The enzyme thus plays a vital role for cysteine and methionine biosynthetic pathways. While bacterial forms of ATPS form tetrameric hetero-dimer complexes which contain a GTPase subunit responsible for GTP hydrolysis and resulting activation of the catalytic reaction, homologues of ATPS from organisms such as yeast and plants exist as monomers or homo-oligomeric complexes and do not require GTP for activation.

 

Reference Protein and Structure

Sequence
P08536 UniProt (2.7.7.4) IPR027535 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1g8f - ATP SULFURYLASE FROM S. CEREVISIAE (1.95 Å) PDBe PDBsum 1g8f
Catalytic CATH Domains
3.40.50.620 CATHdb (see all for 1g8f)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.4)

hydron
CHEBI:15378ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
sulfate
CHEBI:16189ChEBI
→
5'-adenylyl sulfate(2-)
CHEBI:58243ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: ATP sulfurylase, ATP-sulfurylase, Adenosine-5'-triphosphate sulfurylase, Adenosinetriphosphate sulfurylase, Adenylylsulfate pyrophosphorylase, Sulfurylase, Sulfate adenylate transferase,

Enzyme Mechanism

Introduction

ATP sulphurylase catalyses the reaction mostly by simply bringing the substrate in a close proximity and appropriate conformation. The reaction proceeds via a single step nucleophilic substitution without a covalent enzyme bound intermediate being formed.

ATP is able to make an in-line nucleophilic attack on the adjacent sulfate with stereochemical inversion at the alpha-phosphorous leading directly to the formation of APS with pyrophosphate as a leaving group. The pentavalent phosphate intermediate that forms as a result of the nucleophilic attack is stabilised by contact with Arg 290 and Magnesium, but the His-xx-His motif is involved purely in binding and stabilising PPi rather than the general acid-base functionality observed in other members of the family.

Catalytic Residues Roles

UniProt PDB* (1g8f)
His201 His201A Acts as a proton donor to facilitate collapse of the intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg197 (main-N), Thr196, His204, Arg290 Arg197A (main-N), Thr196A, His204A, Arg290A Stabilises the pentacoordinate transition state. hydrogen bond donor, electrostatic stabiliser, steric role
*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 addition, proton transfer, overall reactant used, intermediate formation, bimolecular elimination, intermediate terminated, overall product formed, native state of enzyme regenerated

References

  1. Ullrich TC et al. (2001), EMBO J, 20, 316-329. Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation. DOI:10.1093/emboj/20.3.316. PMID:11157739.
  2. Cleland WW et al. (2006), Chem Rev, 106, 3252-3278. Enzymatic Mechanisms of Phosphate and Sulfate Transfer. DOI:10.1021/cr050287o. PMID:16895327.
  3. Sun M et al. (2005), Biochemistry, 44, 13941-13948. Anatomy of an Energy-Coupling MechanismThe Interlocking Catalytic Cycles of the ATP Sulfurylase−GTPase System†. DOI:10.1021/bi051303e. PMID:16229483.
  4. Lalor DJ et al. (2003), Protein Eng, 16, 1071-1079. Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity. DOI:10.1093/protein/gzg133. PMID:14983089.
  5. Beynon JD et al. (2001), Biochemistry, 40, 14509-14517. Crystal Structure of ATP Sulfurylase from the Bacterial Symbiont of the Hydrothermal Vent TubewormRiftia pachyptila†,‡. DOI:10.1021/bi015643l. PMID:11724564.
  6. Zhang H et al. (1999), J Am Chem Soc, 121, 8692-8697. α-Thio-APS:  A Stereomechanistic Probe of Activated Sulfate Synthesis. DOI:10.1021/ja991648i.
  7. Deyrup AT et al. (1999), J Biol Chem, 274, 28929-28936. Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5'-Phosphosulfate Kinase. DOI:10.1074/jbc.274.41.28929. PMID:10506138.
  8. Venkatachalam KV et al. (1999), J Biol Chem, 274, 2601-2604. Site-selected Mutagenesis of a Conserved Nucleotide Binding HXGH Motif Located in the ATP Sulfurylase Domain of Human Bifunctional 3'-Phosphoadenosine 5'-Phosphosulfate Synthase. DOI:10.1074/jbc.274.5.2601. PMID:9915785.

Step 1. The sulfate substrate attacks the alpha phosphate group, forming a transient penta-coordinate intermediate. Computational modelling of the active site has shown magnesium to bind to the phosphate groups, and kinetic analysis of related enzymes has indicated a magnesium dependent reaction. However, as of yet, the presence of a magnesium ion has not been identified in crystal structures [PMID:11157739]. The hydrogen bond interactions shown to exist between the anionic phosphate groups of ATP and the active site residues by crystallographic data are thought to induce a reactive conformation in the ATP substrate [PMID:11157739].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg197A (main-N) hydrogen bond donor, electrostatic stabiliser, steric role
Thr196A hydrogen bond donor, electrostatic stabiliser, steric role
His201A hydrogen bond donor
His204A electrostatic stabiliser
Arg290A electrostatic stabiliser
His201A proton donor

Chemical Components

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

Step 2. His201 deprotonates the alpha phosphate hydroxyl, initiating the elimination of pyridoxal phosphate and formation of adenyl sulfate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg197A (main-N) hydrogen bond donor, electrostatic stabiliser, steric role
Thr196A hydrogen bond donor, electrostatic stabiliser, steric role
His201A hydrogen bond acceptor
His204A electrostatic stabiliser
Arg290A electrostatic stabiliser
His201A proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The sulfate substrate attacks the alpha phosphate group, forming a transient penta-coordinate intermediate. Computational modelling of the active site has shown magnesium to bind to the phosphate groups, and kinetic analysis of related enzymes has indicated a magnesium dependent reaction. However, as of yet, the presence of a magnesium ion has not been identified in crystal structures [PMID:11157739]. The hydrogen bond interactions shown to exist between the anionic phosphate groups of ATP and the active site residues by crystallographic data are thought to induce a reactive conformation in the ATP substrate [PMID:11157739].

Step 2. His201 deprotonates the alpha phosphate hydroxyl, initiating the elimination of pyridoxal phosphate and formation of adenyl sulfate.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Gary McDowell, Peter Sarkies