Aspartic peptidase

 

There are many different proteins and EC numbers represented by the aspartic endopeptidase family. They are highly conserved and digestive proteases present in vertebrates, fungi, plants, retroviruses, and plant viruses. The catalytic site of pepsin-like enzymes is formed at the junction of the two domains of the protein and contains two catalytic aspartic acid residues, one in each domain. Although they vary widely in their substrate specificity, they all hydrolyse perform the hydrolysis of a peptide chain using a asparate catalytic duo which are found in a neutral and negatively charged state. Aspartate endopeptidases represented here include:

  • Pepsin A, EC:3.4.23.1, shows particularly broad specificity; although bonds involving phenylalanine and leucine are preferred, many others are also cleaved to some extent. Preferential cleavage: hydrophobic, preferably aromatic, residues in P1 and P1' positions. Cleaves 1-Phe-|-Val-2, 4-Gln-|-His-5, 13-Glu-|-Ala-14, 14-Ala-|-Leu-15, 15-Leu-|-Tyr-16, 16-Tyr-|-Leu-17, 23-Gl90-y-|-Phe-24, 24-Phe-|-Phe-25 and 25-Phe-|-Tyr-26 bonds in the B chain of insulin.
  • Gastricsin, EC:3.4.23.3, which shows a more restricted specificity than pepsin A, but shows preferential cleavage at Tyr-|-Xaa bonds. High activity on hemoglobin.
  • Chymosin, EC:3.4.23.4, which is synthesized in the mucosa of the abomasum (fourth stomach) of young (unweaned) ruminants. The enzyme hydrolyzes casein to paracasein. It has broad specificity similar to that of pepsin A. Clots milk by cleavage of a single 104-Ser-Phe-|-Met-Ala-107 bond in kappa-chain of casein. It has historically been use in cheese manufacturing as a milk clotting initiator.
  • Cathepsin D, EC:3.4.23.5, is post-translationally cleaved into two chains (the light and heavy chains). It is active in intracellular protein breakdown. Plays a role in APP processing following cleavage and activation by ADAM30 which leads to APP degradation (PubMed:27333034). Involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer disease and has a specificity similar to, but narrower than, that of pepsin A. Does not cleave the 4-Gln-|-His-5 bond in B chain of insulin.
  • Renin, EC:3.4.23.15, which is known to be a highly specific endopeptidase, whose only known function is to generate angiotensin I from angiotensinogen in the plasma, initiating a cascade of reactions that produce an elevation of blood pressure and increased sodium retention by the kidney.
  • Penicillopepsin, EC:3.4.23.20, which catalyses the hydrolysis of proteins with broad specificity similar to that of pepsin A, preferring hydrophobic residues at P1 and P1', but also cleaving 20-Gly-|-Glu-21 in the B chain of insulin. Clots milk, and activates trypsinogen.
  • Rhizopuspepsin, EC:3.4.23.21, which catalyses the hydrolysis of proteins with broad specificity similar to that of pepsin A, preferring hydrophobic residues at P1 and P1'. Clots milk and activates trypsinogen. Does not cleave 4-Gln-|-His-5, but does cleave 10-His-|-Leu-11 and 12-Val-|-Glu-13 in B chain of insulin.
  • Endothiapepsin, EC:3.4.23.22, which catalyses the hydrolysis of proteins with specificity similar to that of pepsin A, prefers hydrophobic residues at P1 and P1', but does not cleave 14-Ala-|-Leu-15 in the B chain of insulin or Z-Glu-Tyr. Clots milk. Investigation into the inhibition of endothiapepsin has been aimed at forming an inhibitor for renin, another aspartic protease responsible for the formation of the potentially vasoactive peptide angiotensin II.
  • Mucorpepsin, EC:3.4.23.23, which catalyses the hydrolysis of proteins, favoring hydrophobic residues at P1 and P1'. Clots milk. Does not accept Lys at P1, and hence does not activate trypsinogen.
  • Candidapepsin (SAP), EC:3.4.23.24, which acts as a virulence factor within fungal pathogen associated infections. Evidence suggests SAP can degrade proteins associated with immunological and structural defence roles, such and IgG heavy chains, keratin, acidified collagen and extracellular matrix proteins. Its inhibition plays a key role in fighting fungal deceases. It shows preferential cleavage at the carboxyl of hydrophobic amino acids, but fails to cleave 15-Leu-|-Tyr-16, 16-Tyr-|-Leu-17 and 24-Phe-|-Phe-25 of insulin B chain. Activates trypsinogen.
  • Saccharopepsin, EC:3.4.23.25, implicated in the post-translational regulation of S.cerevisiae vacuolar proteinases. Acts on YSCB, on YSCY and on itself. Catalysis the hydrolysis of proteins with broad specificity for peptide bonds. Cleaves -Leu-Leu-|-Val-Tyr- bond in a synthetic substrate. Does not act on esters of Tyr or Arg.
  • Plasmepsin-2, EC:3.4.23.39, which catalyses the hydrolysis of the bonds linking certain hydrophobic residues in hemoglobin or globin. Also cleaves small molecules substrates such as Ala-Leu-Glu-Arg-Thr-Phe-|-Phe(NO2)-Ser-Phe-Pro-Thr.

 

Reference Protein and Structure

Sequence
P56272 UniProt (3.4.23.-) IPR001461 (Sequence Homologues) (PDB Homologues)
Biological species
Gadus morhua (Atlantic cod) Uniprot
PDB
1am5 - THE CRYSTAL STRUCTURE AND PROPOSED AMINO ACID SEQUENCE OF A PEPSIN FROM ATLANTIC COD (GADUS MORHUA) (2.16 Å) PDBe PDBsum 1am5
Catalytic CATH Domains
2.40.70.10 CATHdb (see all for 1am5)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:3.4.23.-)

dipeptide
CHEBI:46761ChEBI
+
water
CHEBI:15377ChEBI
→
alpha-amino acid
CHEBI:33704ChEBI

Enzyme Mechanism

Introduction

The mechanism proceeds via a general acid/base mechanism in which one of the Asp residues is negatively charged (Asp 215) and the other is in a neutral state (Asp 32). Numbering is for the Atlantic cod protein. A low barrier hydrogen bond (LBHB) and peptide dipoles hold the carboxyl groups of the Asps in coplanar conformation with a water molecule between them and hydrogen bonded to both. The resulting symmetrical 10-atom cyclised structure provides a scaffold to impart proximity, orientation and nucleophilicity to the water molecule. Once the substrate binds, a counterclockwise movement of electrons within the cycle occurs, the protons are moved clockwise and the water molecule attacks the carbonyl carbon atom of the amide bond. This generates a tetrahedral intermediate bound to a diprotonated form of the enzyme. The amide group of the tetrahedral intermediate attacks the nearby proton within the ring, causing a clockwise movement of electrons around the cycle, which moves two protons counterclockwise. A zwitterion intermediate is generated, bound to a monoprotonated form of the enzyme. Collapse of the zwitterion cleaves the scissile bond and destroys the coplanarity of the carboxyls. The form is then deprotonated, rehydrated and allowed to restructure the 10-atom cyclic structure with the LBHB.

Catalytic Residues Roles

UniProt PDB* (1am5)
Ser35 Ser35A Forms part of the proton relay chain that reprotonates Asp32 from the conserved active site water molecule. modifies pKa, electrostatic stabiliser
Asp32 Asp32A Acts as a general acid/base, donates a proton to the product and is reprotonated by a conserved active site water, with Ser 35 acting as an intermediary. In the resting state of the enzyme, this residue is in a neutral state. proton acceptor, proton donor
Asp214 Asp215(214)A Acts as a general acid/base, in the resting state of the enzyme this residue is in its negatively charged state. proton acceptor, proton donor
Asn37 (main-C), Tyr75, Trp39 Asn37A (main-C), Tyr75A, Trp39A Activates and correctly positions the conserved water molecule that is responsible for reprotonating Asp 32. modifies pKa, electrostatic stabiliser
Thr217 Thr218(217)A Modifies the pKa of Asp215, activating it and holding it in the negatively charged state. modifies pKa, 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

rate-determining step, proton transfer, bimolecular nucleophilic addition, overall reactant used, bimolecular elimination, overall product formed, native state of enzyme regenerated

References

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Step 1. Asp215 deprotonates the catalytic water molecule, which performs a nucleophilic attack to the carbonyl carbon of the scissile peptide bond. Simultaneously, Asp32 protonates the carbonyl oxygen of the scissile peptide bond, generating a gem-diol intermediate. This molecule possesses two hydroxyl groups bound to the carbon atom of the scissile peptide bond.

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Catalytic Residues Roles

Residue Roles
Ser35A modifies pKa
Asn37A (main-C) modifies pKa
Trp39A modifies pKa
Tyr75A modifies pKa
Thr218(217)A modifies pKa
Asp32A proton donor
Asp215(214)A proton acceptor

Chemical Components

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

Step 2. Asp215 (protonated in the previous step) protonates the amide nitrogen of the scissile peptide bond, which is simultaneously and progressively weakened and elongated.

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Catalytic Residues Roles

Residue Roles
Ser35A electrostatic stabiliser
Asn37A (main-C) electrostatic stabiliser
Trp39A electrostatic stabiliser
Tyr75A electrostatic stabiliser
Thr218(217)A electrostatic stabiliser
Asp215(214)A proton donor

Chemical Components

proton transfer

Step 3. Asp32 deprotonates the hydroxyl group of the second intermediate, concomitantly with deprotonation of the other hydroxyl group by the Asp215 residue.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser35A modifies pKa
Asn37A (main-C) modifies pKa
Trp39A modifies pKa
Tyr75A modifies pKa
Thr218(217)A modifies pKa
Asp32A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp215 deprotonates the catalytic water molecule, which performs a nucleophilic attack to the carbonyl carbon of the scissile peptide bond. Simultaneously, Asp32 protonates the carbonyl oxygen of the scissile peptide bond, generating a gem-diol intermediate. This molecule possesses two hydroxyl groups bound to the carbon atom of the scissile peptide bond.

Step 2. Asp215 (protonated in the previous step) protonates the amide nitrogen of the scissile peptide bond, which is simultaneously and progressively weakened and elongated.

Step 3. Asp32 deprotonates the hydroxyl group of the second intermediate, concomitantly with deprotonation of the other hydroxyl group by the Asp215 residue.

Products.

Contributors

Stuart Lucas, Alex Gutteridge, James W. Murray, Christian Drew, Craig Porter, Steven Smith, Gemma L. Holliday, Marko Babić