Prolidase (Xaa-Pro dipeptidase)

 

Prolidase is a ubiquitously distributed dipeptidase and the only known metalloenzyme in humans capable of cleaving the peptide bond preceding the amino acids proline (Pro) or hydroxyproline (Hyp) in Xaa-Pro (Xaa: any hydrophobic amino acid) dipeptides. Prolidase plays an important role in the degradation of dietary and endogenous proteins to recycle Pro for protein synthesis. Moreover, it's also involved in hydrolysis of Pro- and hydroxyproline-containing dipeptides in the last step of collagen catabolism. Diminished or absent prolidase activity is related to a rare autosomal disease, referred to as prolidase deficiency.

 

Reference Protein and Structure

Sequence
P12955 UniProt (3.4.13.9) IPR000994, IPR007865 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
5m4g - Crystal Structure of Wild-Type Human Prolidase with Mn ions (1.48 Å) PDBe PDBsum 5m4g
Catalytic CATH Domains
(see all for 5m4g)
Cofactors
Manganese(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.4.13.9)

Gly-Pro
CHEBI:70744ChEBI
+
water
CHEBI:15377ChEBI
→
glycine
CHEBI:15428ChEBI
+
proline
CHEBI:26271ChEBI
Alternative enzyme names: Gamma-peptidase, X-Pro dipeptidase, Imidodipeptidase, Peptidase D, Prolidase, Proline dipeptidase,

Enzyme Mechanism

Introduction

A water molecule bound in the active site between the two Mn2+ ions is activated by the abstraction of a proton resulting in the OH- bridged structure. The substrate GlyPro is then bound and His255 moves toward the active site to coordinate the carboxylate group of the substrate. The amide nitrogen and the carbonyl oxygen of the scissile bond of GlyPro are both coordinated by Mn2+ ions facilitating the build-up of a partial positive charge at the carbon atom. The nucleophilic attack of the hydroxide ion results in the formation of a (hypothetical) tetrahedral intermediate. Subsequently, proton transfer and rearrangement of the electrons lead to the breaking of the scissile peptide bond. After the products have left, the productive state of the enzyme is reformed by taking up a water molecule from bulk solvent.

Catalytic Residues Roles

UniProt PDB* (5m4g)
Asp287, Asp276, Glu452, Glu412, His370 Asp287(282)A, Asp276(271)A, Glu452(447)A, Glu412(407)A, His370(365)A Coordinate metal ligand metal ligand
His377 His377(372)A His377 contacts the carbonyl oxygen atom of Gly to stabilize the negative charge built up during the formation of a tetrahedral intermediate. hydrogen bond donor
Arg398 Arg398(393)A Arg398's guanidium group coordinates the C-terminal oxygen atoms of Pro in the Gly-Pro substrate hydrogen bond donor
His255 His255(250)A Upon the formation of closed conformation, His255 moves toward the active site to coordinate the carboxylate group of the substrate. hydrogen bond 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, coordination to a metal ion, bimolecular nucleophilic addition, intermediate formation, overall reactant used, overall product formed, intramolecular rearrangement, unimolecular elimination by the conjugate base, inferred reaction step, native state of enzyme regenerated

References

  1. Wilk P et al. (2017), FEBS J, 284, 2870-2885. Substrate specificity and reaction mechanism of human prolidase. DOI:10.1111/febs.14158. PMID:28677335.
  2. Yang L et al. (2016), Oncotarget, 7, 42340-42352. Dual inhibition of ErbB1 and ErbB2 in cancer by recombinant human prolidase mutant hPEPD-G278D. DOI:10.18632/oncotarget.9851. PMID:27286447.
  3. Alberto ME et al. (2011), Inorg Chem, 50, 3394-3403. Can human prolidase enzyme use different metals for full catalytic activity? DOI:10.1021/ic1022517. PMID:21425789.
  4. Lowther WT et al. (2002), Chem Rev, 102, 4581-4608. Metalloaminopeptidases:  Common Functional Themes in Disparate Structural Surroundings. DOI:10.1021/cr0101757.

Step 1. Two Mn2+ ions are coordinated by the many residues and a water molecule in the active site. Glu412 abstracts a proton from the bridging water molecule, resulting in the OH- -bridged structure

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp287(282)A metal ligand
Asp276(271)A metal ligand
Glu452(447)A metal ligand
Glu412(407)A metal ligand
His370(365)A metal ligand
His377(372)A hydrogen bond donor
Arg398(393)A hydrogen bond donor
His255(250)A hydrogen bond donor
Glu412(407)A proton acceptor

Chemical Components

proton transfer, coordination to a metal ion

Step 2. Nucleophilic attack of the hydroxide ion onto the carbonyl C-atom, leading to the formation of a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp287(282)A metal ligand
Asp276(271)A metal ligand
Glu452(447)A metal ligand
Glu412(407)A metal ligand
His370(365)A metal ligand

Chemical Components

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

Step 3. The intermediate collapses leading to the formation of the products. The nitrogen of the proline is here inferred to be protonated directly by Glu412. The first product (Gly) leaves the active site, then, the return of the protein conformation to the open state kicks the second product (Pro) out of the active site. After the products have left, the productive state of the enzyme is reformed by taking up a water molecule from bulk solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp287(282)A metal ligand
Asp276(271)A metal ligand
Glu452(447)A metal ligand
Glu412(407)A metal ligand
His370(365)A metal ligand
Glu412(407)A proton donor

Chemical Components

overall product formed, proton transfer, intramolecular rearrangement, ingold: unimolecular elimination by the conjugate base, inferred reaction step
Download: Image, Marvin File

Step 1. Two Mn2+ ions are coordinated by the many residues and a water molecule in the active site. Glu412 abstracts a proton from the bridging water molecule, resulting in the OH- -bridged structure

Step 2. Nucleophilic attack of the hydroxide ion onto the carbonyl C-atom, leading to the formation of a tetrahedral intermediate.

Step 3. The intermediate collapses leading to the formation of the products. The nitrogen of the proline is here inferred to be protonated directly by Glu412. The first product (Gly) leaves the active site, then, the return of the protein conformation to the open state kicks the second product (Pro) out of the active site. After the products have left, the productive state of the enzyme is reformed by taking up a water molecule from bulk solvent.

Products.

Contributors

Trung Nguyen, Antonio Ribeiro