Bacterial leucyl aminopeptidase

 

Aminopeptidases catalyse the hydrolysis of a wide range of N-terminal amino acid residues from proteins and polypeptides. These enzymes have a broad substrate specificity and are widely distributed in bacteria, yeast, plant and animal tissues. Functions of aminopeptidases include protein maturation, protein degradation, hormone level regulation and cell cycle control. Since aminopeptidase activity has been reported on the surface of tumour cells, they are very likely key players in tumour growth and metastatic proliferation.

 

Reference Protein and Structure

Sequence
Q01693 UniProt (3.4.11.10) IPR012189 (Sequence Homologues) (PDB Homologues)
Biological species
Vibrio proteolyticus (Bacteria) Uniprot
PDB
1lok - The 1.20 Angstrom Resolution Crystal Structure of the Aminopeptidase from Aeromonas proteolytica Complexed with Tris: A Tale of Buffer Inhibition (1.2 Å) PDBe PDBsum 1lok
Catalytic CATH Domains
3.40.630.10 CATHdb (see all for 1lok)
Cofactors
Zinc(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.4.11.10)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Aeromonas proteolytica aminopeptidase,

Enzyme Mechanism

Introduction

A zinc-activated water molecule, deprotonated by glutamate, attacks the carbonyl carbon of the substrate. The tetrahedral oxyanion transition state is stabilised by one of the zinc ions. The transition state collapses, breaking the peptide bond, and the leaving group is protonated by the glutamate residue, returning the enzyme to its native state.

Catalytic Residues Roles

UniProt PDB* (1lok)
Glu257 Glu151A Deprotonates the nucleophilic water molecule, protonates the leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp223 Asp117A Binds both the zinc ions. metal ligand
Asp285, His203 Asp179A, His97A Forms part of the zinc 1 binding site. metal ligand
His362, Glu258 His256A, Glu152A Forms part of the zinc 2 binding site. metal ligand
*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, overall reactant used, intermediate formation, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, intermediate terminated, intermediate collapse, overall product formed, native state of enzyme regenerated, rate-determining step

References

  1. Chen G et al. (1997), Biochemistry, 36, 4278-4286. Mechanistic Studies on the Aminopeptidase fromAeromonas proteolytica:  A Two-Metal Ion Mechanism for Peptide Hydrolysis†. DOI:10.1021/bi9618676. PMID:9100023.
  2. Chen SL et al. (2008), J Phys Chem B, 112, 2494-2500. Peptide hydrolysis by the binuclear zinc enzyme aminopeptidase from Aeromonas proteolytica: a density functional theory study. DOI:10.1021/jp710035j. PMID:18247603.
  3. Desmarais W et al. (2006), J Biol Inorg Chem, 11, 398-408. The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica. DOI:10.1007/s00775-006-0093-x. PMID:16596389.
  4. Desmarais WT et al. (2002), Structure, 10, 1063-1072. The 1.20 A resolution crystal structure of the aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition. DOI:10.2210/pdb1lok/pdb. PMID:12176384.
  5. Stamper C et al. (2001), Biochemistry, 40, 7035-7046. Inhibition of the Aminopeptidase fromAeromonas proteolyticabyl-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis†. DOI:10.1021/bi0100891. PMID:11401547.

Step 1. Glu151 deprotonates the zinc activated water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp117A metal ligand
His97A metal ligand
Glu152A metal ligand
His256A metal ligand
Asp179A metal ligand
Glu151A hydrogen bond acceptor, proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The activated hydroxide attacks the peptide carbonyl in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp117A metal ligand
His97A metal ligand
Glu152A metal ligand
His256A metal ligand
Asp179A metal ligand
Glu151A hydrogen bond donor

Chemical Components

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

Step 3. The oxyanion initiates an elimination that cleaves the peptide bond and releases the protein's new N-terminal amino acid, which protonates from Glu151, and the amino acid product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His97A metal ligand
Asp117A metal ligand
Glu152A metal ligand
Asp179A metal ligand
His256A metal ligand
Glu151A hydrogen bond donor, hydrogen bond acceptor, proton donor

Chemical Components

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

Step 1. Glu151 deprotonates the zinc activated water molecule.

Step 2. The activated hydroxide attacks the peptide carbonyl in a nucleophilic addition.

Step 3. The oxyanion initiates an elimination that cleaves the peptide bond and releases the protein's new N-terminal amino acid, which protonates from Glu151, and the amino acid product.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Stuart Lucas, Craig Porter