E1 ubiquitin-activating enzyme

 

Ubiquitination is mediated by three enzymes, E1 (PDB:3cmm), E2 (PDB:1ayz) and E3 (PDB:1c4z). The first, E1, is essential for ubiquitin activation and transferring the substrate onto the second cascade enzyme, E2, which responsible for mediating repeated ubiquitination at the eventual substrate, which is brought into proximity of E2 by the active site of E3, the enzyme which also regulates substrate specificity. This cascade regulates the ubiquitination of specific proteins, generating post-translational modifications that activate a number of possible cellular responses, depending upon the site and type of ubiquitin marker.

This entry represents the first of the three reactions occurring in this cascade: ubiquitin:[E1 ubiquitin-activating enzyme] ligase (AMP-forming). E1 catalyses the ATP-dependent activation of ubiquitin through the formation of a thioester bond between the C-terminal glycine of ubiquitin and the sulfhydryl side group of a cysteine residue in the E1 protein. Several E1 type enzymes have been identified to catalyse substrate-specific transfer reactions for ubiquitin-like proteins, although a number of these enzymes will also catalyse the transfer of ubiquitin and other ubiquitin-like proteins.

 

Reference Protein and Structure

Sequences
P22515 UniProt (6.2.1.45)
P06104 UniProt (2.3.2.23) IPR018075 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
3cmm - Crystal Structure of the Uba1-Ubiquitin Complex (2.7 Å) PDBe PDBsum 3cmm
Catalytic CATH Domains
3.50.50.80 CATHdb 1.10.10.2660 CATHdb 3.40.50.720 CATHdb (see all for 3cmm)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.2.1.45)

L-cysteine residue
CHEBI:29950ChEBI
+
carboxylic acid anion
CHEBI:29067ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
S-(long-chain fatty acyl)-L-cysteine residue
CHEBI:133479ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
diphosphate(4-)
CHEBI:18361ChEBI
+
5'-acylphosphoadenosine(1-)
CHEBI:60043ChEBI
Alternative enzyme names: Ubiquitin activating enzyme, E1, Ubiquitin-activating enzyme E1,

Enzyme Mechanism

Introduction

The two-step reaction consists of the ATP-dependent formation of an E1-ubiquitin adenylate intermediate in which the C-terminal glycine of ubiquitin is bound to AMP via an acyl-phosphate linkage, then followed by the conversion to an E1-ubiquitin thioester bond via the cysteine residue on E1 in the second step.

Catalytic Residues Roles

UniProt PDB* (3cmm)
Asn781 (main-N), Asp782 (main-N) Asn781(772)A (main-N), Asp782(773)A (main-N) Forms the oxyanion hole that stabilises the negatively charged intermediates and transition states involved in the transfer of the ubiquitin from AMP to Cys600A in E1. hydrogen bond donor, electrostatic stabiliser
Thr601 Thr601(592)A Structural conservation and crystallographic data suggests that Thr601A may act as a general base, both towards Cys600A of E1 and also Cys88A of E2. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, increase acidity, increase nucleophilicity
Asp544 Asp544(535)A Helps ensure the substrates are held in the correct positions for the reaction to occur, also destabilises the ground state during the ubiquitin adenylation steps. steric role
Cys600 Cys600(591)A This is the E1 residue that becomes ubiqutinated. Also acts as a general acid/base in this portion of the reaction. covalently attached, hydrogen bond donor, nucleophile, proton donor, activator
Arg481, Arg603, Arg21 Arg481(472)A, Arg603(594)A, Arg21(12)A Helps stabilise the reactive intermediates formed during the course of the reaction. hydrogen bond donor, steric role, 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

bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation, bimolecular nucleophilic addition, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, intermediate collapse

References

  1. Schulman BA et al. (2009), Nat Rev Mol Cell Biol, 10, 319-331. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. DOI:10.1038/nrm2673. PMID:19352404.
  2. Olsen SK et al. (2010), Nature, 463, 906-912. Active site remodelling accompanies thioester bond formation in the SUMO E1. DOI:10.1038/nature08765. PMID:20164921.
  3. Lee I et al. (2008), Cell, 134, 268-278. Structural Insights into E1-Catalyzed Ubiquitin Activation and Transfer to Conjugating Enzymes. DOI:10.1016/j.cell.2008.05.046. PMID:18662542.
  4. Capili AD et al. (2007), Curr Opin Struct Biol, 17, 726-735. Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways. DOI:10.1016/j.sbi.2007.08.018. PMID:17919899.
  5. Passmore LA et al. (2004), Biochem J, 379, 513-525. Getting into position: the catalytic mechanisms of protein ubiquitylation. DOI:10.1042/bj20040198. PMID:14998368.
  6. Pickart CM et al. (2004), Biochim Biophys Acta, 1695, 55-72. Ubiquitin: structures, functions, mechanisms. DOI:10.1016/j.bbamcr.2004.09.019. PMID:15571809.
  7. Wu PY et al. (2003), EMBO J, 22, 5241-5250. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. DOI:10.1093/emboj/cdg501. PMID:14517261.
  8. Lake MW et al. (2001), Nature, 414, 325-329. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. DOI:10.1038/35104586. PMID:11713534.
  9. Scheffner M et al. (1995), Nature, 373, 81-83. Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. DOI:10.1038/373081a0. PMID:7800044.

Step 1. The catalytic residues in the adenylation site are thought to induce a reactive conformation in ATP, promoting nucleophilic attack from the ubiquitin C terminus, as well as stabilising the pentavalent transition state [PMID:19352404, PMID:15571809]. The C terminus of ubiquitin attacks the alpha phosphate of ATP within the adenylation binding site.

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Catalytic Residues Roles

Residue Roles
Arg481(472)A steric role, hydrogen bond donor, electrostatic stabiliser
Arg21(12)A steric role, hydrogen bond donor, electrostatic stabiliser
Asp544(535)A steric role

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation

Step 2. The adenylation site is approx. 35 A from the catalytic cysteine, situated in the thio-ester formation site. A change in conformation upon adenylation is thought to bring the two reaction sites closer. This structural rearrangement is also thought to orientate residues into a catalytically active arrangement, including the postulated general base [PMID:19352404] that activates Cys600A towards nucleophilic attack at the adenylated ubiquitin.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg603(594)A hydrogen bond donor, electrostatic stabiliser
Thr601(592)A increase acidity, increase nucleophilicity, hydrogen bond acceptor
Asp782(773)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn781(772)A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys600(591)A hydrogen bond donor
Thr601(592)A proton acceptor
Cys600(591)A proton donor
Thr601(592)A proton donor
Cys600(591)A nucleophile
Thr601(592)A proton relay

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, enzyme-substrate complex formation

Step 3. The tetrahedral anionic intermediate collapses, releasing AMP and forming the enzyme-substrate adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg603(594)A hydrogen bond donor, electrostatic stabiliser
Thr601(592)A hydrogen bond acceptor, hydrogen bond donor
Asp782(773)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn781(772)A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys600(591)A covalently attached, activator

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation

Step 4. A second round of ubiquitin adenylation is coupled to the formation of the high energy, activated enzyme bound ubiquitin intermediates. The C terminus of a second ubiquitin attacks the alpha phosphate of ATP within the adenylation binding site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg481(472)A steric role, hydrogen bond donor, electrostatic stabiliser
Arg21(12)A steric role, hydrogen bond donor, electrostatic stabiliser
Asp544(535)A steric role

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation
Download: Image, Marvin File

Step 1. The catalytic residues in the adenylation site are thought to induce a reactive conformation in ATP, promoting nucleophilic attack from the ubiquitin C terminus, as well as stabilising the pentavalent transition state [PMID:19352404, PMID:15571809]. The C terminus of ubiquitin attacks the alpha phosphate of ATP within the adenylation binding site.

Step 2. The adenylation site is approx. 35 A from the catalytic cysteine, situated in the thio-ester formation site. A change in conformation upon adenylation is thought to bring the two reaction sites closer. This structural rearrangement is also thought to orientate residues into a catalytically active arrangement, including the postulated general base [PMID:19352404] that activates Cys600A towards nucleophilic attack at the adenylated ubiquitin.

Step 3. The tetrahedral anionic intermediate collapses, releasing AMP and forming the enzyme-substrate adduct.

Step 4. A second round of ubiquitin adenylation is coupled to the formation of the high energy, activated enzyme bound ubiquitin intermediates. The C terminus of a second ubiquitin attacks the alpha phosphate of ATP within the adenylation binding site.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday