Xaa-Pro aminopeptidase

 

Aminopeptidase P cleaves an Xaa-Pro bond at the N-terminal of a polypeptide (even a dipeptide or tripeptide), where Xaa can be any amino acid. This substrate specificity is important as many biologically active peptides have Xaa-Pro at the N-terminus.

This protein is part of the peptidase M24B family.

 

Reference Protein and Structure

Sequence
P15034 UniProt (3.4.11.9) IPR001714 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1a16 - AMINOPEPTIDASE P FROM E. COLI WITH THE INHIBITOR PRO-LEU (2.3 Å) PDBe PDBsum 1a16
Catalytic CATH Domains
3.90.230.10 CATHdb 3.40.350.10 CATHdb (see all for 1a16)
Cofactors
Manganese(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.4.11.9)

Ala-Pro
CHEBI:73393ChEBI
+
water
CHEBI:15377ChEBI
→
L-alanine
CHEBI:16977ChEBI
+
L-proline
CHEBI:17203ChEBI
Alternative enzyme names: X-Pro aminopeptidase, Aminoacylproline aminopeptidase, Aminopeptidase P, Proline aminopeptidase,

Enzyme Mechanism

Introduction

A hydroxide ion complexed between two manganese (II) ions is the nucleophile for this reaction. It is further activated by a hydrogen bond to glutamate. The nucleophile attacks the carbonyl of the substrate, and the negatively charged tetrahedral intermediate is stabilised by an oxyanion hole. When the intermediate collapses the peptide bond is broken and the products (the C-terminal peptide and the N-terminal amino acid) are released. A solvent water molecule enters the active site to replace the original hydroxide ion.

Catalytic Residues Roles

UniProt PDB* (1a16)
His244 His243A Stabilizes substrate binding, may also be part of a proton shuttle based on similarity to arginase. electrostatic stabiliser
His351 His350A Forms part of a hydrophobic binding pocket that gives the enzyme its proline specificity, may also be part of a proton shuttle based on similarity to arginase. electrostatic stabiliser
His362 His361A Stabilizes substrate binding and is also part of the oxyanion hole that stabilises the negative charge on the gem-diol catalytic intermediate. Also suggested as part of a proton shuttle. electrostatic stabiliser
Glu384 Glu383A Has an essential role in metal binding in the presence of substrate. Also activates the water molecule to act as a nucleophile. activator, proton shuttle (general acid/base), metal ligand
Asp261, Asp272, Glu407 Asp260A, Asp271A, Glu406A Forms the second Mn(II) binding site (site numbering from UniProtKB entry) proton shuttle (general acid/base), metal ligand
Asp272, Glu407, His355 Asp271A, Glu406A, His354A Forms the first Mn(II) binding site (site numbering from UniProtKB entry) metal ligand
Arg405, Tyr388 Arg404A, Tyr387A Part of a hydrogen bond network (Asp260 - Arg404 - Tyr387). This network is responsible for one of the proton relays that occurs in the active site. proton shuttle (general acid/base)
Asp39 Asp38A(AC) activates and stabilises His361. activator, 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

References

  1. Graham SC et al. (2006), Biochemistry, 45, 964-975. Kinetic and Crystallographic Analysis of MutantEscherichia coliAminopeptidase P:  Insights into Substrate Recognition and the Mechanism of Catalysis†. DOI:10.1021/bi0518904. PMID:16411772.
  2. Graham SC et al. (2008), Arch Biochem Biophys, 469, 200-208. Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu. DOI:10.1016/j.abb.2007.10.009. PMID:17983589.
  3. Jao SC et al. (2006), Biochemistry, 45, 1547-1553. Tyrosine 387 and Arginine 404 Are Critical in the Hydrolytic Mechanism ofEscherichia coliAminopeptidase P†. DOI:10.1021/bi051786m. PMID:16460001.
  4. Graham SC et al. (2004), Acta Crystallogr D Biol Crystallogr, 60, 1770-1779. Structure ofEscherichia coliaminopeptidase P in complex with the inhibitor apstatin. DOI:10.1107/s0907444904018724. PMID:15388923.
  5. Lowther WT et al. (2002), Chem Rev, 102, 4581-4608. Metalloaminopeptidases:  Common Functional Themes in Disparate Structural Surroundings. DOI:10.1021/cr0101757.
  6. Wilce MC et al. (1998), Proc Natl Acad Sci U S A, 95, 3472-3477. Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli. DOI:10.1073/pnas.95.7.3472. PMID:9520390.
  7. Kanyo ZF et al. (1996), Nature, 383, 554-557. Structure of a unique binuclear manganese cluster in arginase. DOI:10.1038/383554a0. PMID:8849731.

Step 1.

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Catalytic Residues Roles

Residue Roles
Asp38A(AC) electrostatic stabiliser, activator
His350A electrostatic stabiliser
His361A electrostatic stabiliser
Asp260A proton shuttle (general acid/base)
Tyr387A proton shuttle (general acid/base)
Arg404A proton shuttle (general acid/base)
Asp271A metal ligand
His354A metal ligand
Glu383A metal ligand
Glu406A metal ligand
Asp260A metal ligand
Glu383A activator, proton shuttle (general acid/base)
His243A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Emma LR Compton, Craig Porter, Gemma L. Holliday