Pyroglutamyl-peptidase I

 

Pyroglutamyl peptidase I is a cytosolic cysteine protease that cleaves cleaves N-terminal pyroglutamyl (pGlu) residues from a broad specturm of pGlu-containing peptides including thyrotropin releasing hormone (TRH), luterinizing hormone releasing hormone (LH-RH), bombesin (from the skin of reptiles), neurotensin (from mammalian brain) and the gastrins (gastric secretion stimulants). Such peptides with an N-terminal pGlu residue are resistant to degradation by conventional aminopeptidases. In the case of TRH and LH-RH this is important in protecting the hormones during their journey from the hypothalamus (where they are produced) to the pituitary gland. Pyroglutamyl peptidase I is widely found in bacteria and plants as well as in animals; in archaea and eubacteria it may have a role in detoxification/nutrient assimilation of N-terminally blocked peptides.

 

Reference Protein and Structure

Sequence
P46107 UniProt (3.4.19.3) IPR029762 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus amyloliquefaciens (Bacteria) Uniprot
PDB
1aug - CRYSTAL STRUCTURE OF THE PYROGLUTAMYL PEPTIDASE I FROM BACILLUS AMYLOLIQUEFACIENS (2.0 Å) PDBe PDBsum 1aug
Catalytic CATH Domains
3.40.630.20 CATHdb (see all for 1aug)
Click To Show Structure

Enzyme Reaction (EC:3.4.19.3)

N-terminal 5-oxo-L-proline residue
CHEBI:87215ChEBI
+
water
CHEBI:15377ChEBI
→
H group
CHEBI:64428ChEBI
+
5-oxo-D-prolinate
CHEBI:57948ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 5-oxoprolyl-peptidase, L-pyroglutamyl peptide hydrolase, L-pyrrolidonecarboxylate peptidase, Pyrase, Pyroglutamate aminopeptidase, Pyroglutamidase, Pyroglutamyl aminopeptidase, Pyrrolidone-carboxyl peptidase, Pyrrolidonecarboxylyl peptidase, Pyrrolidonyl peptidase, Pyrrolidone-carboxylate peptidase, PYRase, Pyrrolidone carboxyl peptidase,

Enzyme Mechanism

Introduction

Pyroglutamyl peptidase I uses a Cys-His-Asp triad to catalyse peptide bond cleavage. Cys 144 acts as a nucleophile to attack the peptide carbonyl, while His 168 deprotonates the nucleophilic cysteine and protonates the departing leaving amine leaving group of the substrate. Later it deprotonates the water molecule that hydrolyses the acyl enzyme intermediate. Its pKa value is modified by the nearby Asp 81. Pyroglutamyl peptidase I does not a conventional oxyanion hole of two backbone NH groups; instead the tetrahedral intermediate is thought to be stabilised by the guanidinium group of Arg 91 as well as by the backbone NH of Cys 144.

Catalytic Residues Roles

UniProt PDB* (1aug)
Glu81 Glu81A Modifies pKa of His 168. electrostatic stabiliser
His168 His168A Deprotonates the nucleophilic Cys 144. Protonates the departing amine leaving group of the tetrahedral intermediate. Deprotonates the water molecule that attacks the acyl-enzyme intermediate. proton acceptor, proton donor
Cys144, Cys144 (main-N) Cys144A, Cys144A (main-N) Acts as a nucleophile to attack the peptide bond. Backbone NH forms part of the oxyanion hole that stabilises the tetrahedral intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
Arg91 Arg91A Side chain guanidinium group forms part of the oxyanion hole that stabilises the tetrahedral intermediate. 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, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Odagaki Y et al. (1999), Structure, 7, 399-411. The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease. DOI:10.1016/s0969-2126(99)80053-7. PMID:10196127.
  2. Kamiya K et al. (2010), J Phys Chem B, 114, 6567-6578. Energy compensation mechanism for charge-separated protonation states in aspartate-histidine amino acid residue pairs. DOI:10.1021/jp906148m. PMID:20411975.
  3. Dando PM et al. (2003), Protein Expr Purif, 28, 111-119. Pyroglutamyl-peptidase I: cloning, sequencing, and characterisation of the recombinant human enzyme. DOI:10.1016/S1046-5928(02)00632-0.
  4. Ito K et al. (2001), J Biol Chem, 276, 18557-18562. The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis. DOI:10.1074/jbc.M011724200. PMID:11359794.
  5. Le Saux O et al. (1996), J Bacteriol, 178, 3308-3313. Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase. PMID:8655512.
  6. Yoshimoto T et al. (1993), J Biochem, 113, 67-73. Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme. PMID:8095933.

Step 1. His168 deprotonates Cys144 which activates it to attack the carbon of the carbonyl bond of the peptide bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu81A electrostatic stabiliser
Cys144A (main-N) electrostatic stabiliser
Arg91A electrostatic stabiliser
His168A proton acceptor
Cys144A nucleophile, proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used, rate-determining step

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from His168.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu81A electrostatic stabiliser
Arg91A electrostatic stabiliser
Cys144A (main-N) electrostatic stabiliser
His168A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed

Step 3. His168 abstracts a proton from a water which activates it so that it can attack the carbon of the thioester bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu81A electrostatic stabiliser
Arg91A electrostatic stabiliser
Cys144A (main-N) electrostatic stabiliser
His168A proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates another elimination and this results in the cleavage of the thioester bond. The released Cys144 can now accept a proton from His168 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu81A electrostatic stabiliser
Arg91A electrostatic stabiliser
Cys144A (main-N) electrostatic stabiliser
Cys144A proton acceptor, nucleofuge
His168A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His168 deprotonates Cys144 which activates it to attack the carbon of the carbonyl bond of the peptide bond in a nucleophilic addition.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from His168.

Step 3. His168 abstracts a proton from a water which activates it so that it can attack the carbon of the thioester bond in a nucleophilic addition.

Step 4. The oxyanion initiates another elimination and this results in the cleavage of the thioester bond. The released Cys144 can now accept a proton from His168 which returns the enzyme to its native state.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby