Neprilysin

 

Neutral endopeptidase, or neprelysin, is a Zinc protease located in the membrane of mammalian tissue and responsible for the hydrolysis of peptides such as Insulin at the cell surface. As a result it plays a key role in many of the processes involved in cell signalling. Although it displays little sequence or structural homology to other known Zinc dependent proteases such as the bacterial protein thermolysin, it's active site can be successfully aligned to the active site of thermolysin, thus convergent evolution is believed to account for the similarity in mechanism between the two that is observed.

 

Reference Protein and Structure

Sequence
P08473 UniProt (3.4.24.11) IPR029727 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1r1j - STRUCTURAL ANALYSIS OF NEPRILYSIN WITH VARIOUS SPECIFIC AND POTENT INHIBITORS (2.35 Å) PDBe PDBsum 1r1j
Catalytic CATH Domains
3.40.390.10 CATHdb (see all for 1r1j)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.4.24.11)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: CALLA, CALLA (common acute lymphoblastic leukemia-associated) antigens, CALLA antigen, CALLA glycoprotein, CALLA glycoproteins, CD10, NEP, Acute lymphoblastic leukemia antigen, Common acute lymphoblastic leukemia antigen, Common acute lymphoblastic leukemia-associated antigens, Endopeptidase, Endopeptidase 24.11, Endopeptidase-2, Enkephalinase, Kidney-brush-border neutral endopeptidase, Kidney-brush-border neutral peptidase, Kidney-brush-border neutral proteinase, Membrane metalloendopeptidase, Neutral endopeptidase, Neutral endopeptidase 24.11, Neutral metallendopeptidase,

Enzyme Mechanism

Introduction

The Zinc ion and the catalytic residue Glu 584 act together to activate a water molecule for nucleophilic attack on the peptide substrate. This forms an oxyanion intermediate, stabilised by His 711 in a hydrogen bond network involving Asp 650 and Arg 717. Subsequent collapse of the intermediate results in cleavage of the peptide bond and release of the products.

Catalytic Residues Roles

UniProt PDB* (1r1j)
Glu585 Glu584(531)A Acts as a general base to activate water for nucleophilic attack of peptide bond proton shuttle (general acid/base)
His588, Glu647, His584 His587(534)A, Glu646(593)A, His583(530)A Form Zinc binding site of enzyme metal ligand
Asp651 Asp650(597)A Forms Hydrogen bond network to raise the pKa of His 711, thus allowing it to exist in the protonated form at physiological pH so it can stabilise the tetrahedral intermediate. electrostatic stabiliser
His712 His711(658)A Forms hydrogen bond to the oxyanion thus stabilising the tetrahedral intermediate in the reaction. electrostatic stabiliser
Arg718 Arg717(664)A Hydrogen bonds to His 711 to raise its pKa so that it remains protonated at physiological pH. 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. Devault A et al. (1988), FEBS Lett, 231, 54-58. Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis Histidine residues 583 and 587 are essential for catalysis. DOI:10.1016/0014-5793(88)80701-4. PMID:3162886.
  2. Oefner C et al. (2000), J Mol Biol, 296, 341-349. Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon. DOI:10.1006/jmbi.1999.3492. PMID:10669592.
  3. Marie-Claire C et al. (1997), Biochemistry, 36, 13938-13945. Evidence by Site-Directed Mutagenesis That Arginine 203 of Thermolysin and Arginine 717 of Neprilysin (Neutral Endopeptidase) Play Equivalent Critical Roles in Substrate Hydrolysis and Inhibitor Binding. DOI:10.1021/bi9712495. PMID:9374873.
  4. Le Moual H et al. (1994), Eur J Biochem, 221, 475-480. Asp650 is crucial for catalytic activity of neutral endopeptidase 24-11. DOI:10.1111/j.1432-1033.1994.tb18760.x. PMID:8168535.
  5. Dion N et al. (1993), FEBS Lett, 318, 301-304. Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. DOI:10.1016/0014-5793(93)80533-z. PMID:8440386.
  6. Devault A et al. (1988), J Biol Chem, 263, 4033-4040. Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site. PMID:2894375.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg717(664)A electrostatic stabiliser
His711(658)A electrostatic stabiliser
Asp650(597)A electrostatic stabiliser
His583(530)A metal ligand
His587(534)A metal ligand
Glu646(593)A metal ligand
Glu584(531)A proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Hannah Gilbert, Peter Sarkies, Gemma L. Holliday, Charity Hornby