Protein-synthesizing GTPase (elongation factor Tu)

 

Elongation factor Tu hydrolyses GTP to give GDP and Pi, thus providing the free energy that the ribosome needs to incorporate amino acids into the growing polypeptide. As such, it shows homology to other GTP binding proteins such as the oncogene ras, but may show a different mechanism of GTP hydrolysis. Many antibiotics are able to inhibit EFTu, making study of its mechanism particularly useful in view of the possibility of synthesising new antibiotics.

 

Reference Protein and Structure

Sequences
P0CE47 UniProt
Q7M0J8 UniProt IPR004541 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
5jbq - EF-TU (ESCHERICHIA COLI) IN COMPLEX WITH THIOMURACIN ANALOG (2.006 Å) PDBe PDBsum 5jbq
Catalytic CATH Domains
3.40.50.300 CATHdb (see all for 5jbq)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.6.5.3)

GTP(4-)
CHEBI:37565ChEBI
+
water
CHEBI:15377ChEBI
→
GDP(3-)
CHEBI:58189ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: Elongation factor (EF), Initiation factor (IF), Peptide-release or termination factor, Peptide-release factor (RF),

Enzyme Mechanism

Introduction

The reaction of the GTPase of EF-Tu follows a concerted early proton transfer to the γ-phosphate, resulting in subsequent nucleophilic attack on the γ-phosphate monoanion by hydroxide. His 85 is shown to be doubly protonated and is involved in the stabilization of the negative charge development along with the back bone NH of Gly 84 which together form an oxyanion hole. This effect also requires the effect of Asp 22 which replies the negative charge of the phosphate toward His 85. Lys 25 lastly stabilises the leaving group phosphate's negative charge.

Catalytic Residues Roles

UniProt PDB* (5jbq)
Asp22 Asp21(22)A Repels the phosphate's negative charge to the stabilising positive Histidine. electrostatic stabiliser
Thr26 Thr25(26)A Forms the Mg2+ binding site metal ligand
Gly84 (main-N), His85 Gly83(84)A (main-N), His84(85)A Forms the oxyanion hole and stabilises the negative charge of the transition state through hydrogen bonding. electrostatic stabiliser
Lys25 Lys24(25)A Stabilises the phosphate leaving group's negative charge. 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, bimolecular nucleophilic addition, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

References

  1. Geggier P et al. (2010), J Mol Biol, 399, 576-595. Conformational Sampling of Aminoacyl-tRNA during Selection on the Bacterial Ribosome. DOI:10.1016/j.jmb.2010.04.038. PMID:20434456.
  2. Åqvist J et al. (2015), Biochemistry, 54, 546-556. The conformation of a catalytic loop is central to GTPase activity on the ribosome. DOI:10.1021/bi501373g. PMID:25515218.
  3. Wallin G et al. (2013), Nat Commun, 4, 1733-. Energetics of activation of GTP hydrolysis on the ribosome. DOI:10.1038/ncomms2741. PMID:23591900.
  4. Aleksandrov A et al. (2013), RNA, 19, 1218-1225. Mechanism of activation of elongation factor Tu by ribosome: catalytic histidine activates GTP by protonation. DOI:10.1261/rna.040097.113. PMID:23864225.
  5. B RP et al. (2013), Proc Natl Acad Sci U S A, 110, 20509-20514. Quantitative exploration of the molecular origin of the activation of GTPase. DOI:10.1073/pnas.1319854110. PMID:24282301.

Step 1. The water protonates the gamma phosphate and the hydroxide formed then nucleophilically attacks the phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly83(84)A (main-N) electrostatic stabiliser
Asp21(22)A electrostatic stabiliser
Lys24(25)A electrostatic stabiliser
His84(85)A electrostatic stabiliser
Thr25(26)A metal ligand

Chemical Components

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

Step 2. The pentacoordinate transition state collapse resulting in the cleavage of the phosphodiester bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly83(84)A (main-N) electrostatic stabiliser
Asp21(22)A electrostatic stabiliser
Lys24(25)A electrostatic stabiliser
His84(85)A electrostatic stabiliser
Thr25(26)A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed
Download: Image, Marvin File

Step 1. The water protonates the gamma phosphate and the hydroxide formed then nucleophilically attacks the phosphate.

Step 2. The pentacoordinate transition state collapse resulting in the cleavage of the phosphodiester bond.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Charity Hornby