Carboxypeptidase D

 

Carboxypeptidase D is a serine protease which specifically removes basic or hydrophobic residues from the C-terminus of the substrate protein.

Carboxypeptidase D is a member of the alpha beta hydrolase family and contains a Ser-His-Asp catalytic triad typical of the family.

In humans, mutations arising in this enzyme result in genetic disorders such as galactosialidosis, a disease associated with incorrect protein degradation in the lysosome.

Carboxypeptidase D from yeast and wheat have had their structures determined, The wheat catalytic triad is made up of residues from both subunits of the homodimer whilst yeast carboxypeptidase D is a monomer, however, both have similar active site geometries.

 

Reference Protein and Structure

Sequence
P08819 UniProt (3.4.16.6) IPR001563 (Sequence Homologues) (PDB Homologues)
Biological species
Triticum aestivum (bread wheat) Uniprot
PDB
1whs - STRUCTURE OF THE COMPLEX OF L-BENZYLSUCCINATE WITH WHEAT SERINE CARBOXYPEPTIDASE II AT 2.0 ANGSTROMS RESOLUTION (2.0 Å) PDBe PDBsum 1whs
Catalytic CATH Domains
3.40.50.1820 CATHdb 3.40.50.11320 CATHdb (see all for 1whs)
Click To Show Structure

Enzyme Reaction (EC:3.4.16.6)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Saccharomyces cerevisiae KEX1 gene product, CPDW-II, KEX1 carboxypeptidase, KEX1 proteinase, KEX1DELTA-P, Carboxypeptidase Kex1, Cereal serine carboxypeptidase II, Gene KEX1 serine carboxypeptidase, Carboxypeptidase S1, Carboxypeptidase KEX1, Serine carboxypeptidase, Phaseolus proteinase,

Enzyme Mechanism

Introduction

Carboxypeptidase D uses a catalytic triad to activate serine 146 as a nucleophile to attack the scissile peptide bond. Histidine 397 and aspartate 338 from the neighbouring subunit complete the triad. The backbone amides of glycine 53 and tyrosine 147 make up the oxyanion hole to stabilise the tetrahedral intermediate.

Catalytic Residues Roles

UniProt PDB* (1whs)
Ser158 Ser146(153)A Acts as a nucleophile in the acylation step. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge
Gly62 (main-N), Tyr159 (main-N) Gly53(57)A (main-N), Tyr147(154)A (main-N) The main chain amide forms part of the oxyanion hole. hydrogen bond donor, electrostatic stabiliser
Asp361 Asp338(75)B Part of the Ser-His-Asp triad that activates Ser146 for nucleophilic attack. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
His413 His397(127)B Part of the Ser-His-Asp triad that activates Ser146 for nucleophilic attack. His397 acts as a general acid/base, deprotonating Ser146 and furnishes the proton to the first product. It then deprotonates water for the step in which Ser146 is regenerated. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, 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

proton transfer, bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, intermediate collapse, native state of enzyme regenerated, intermediate terminated

References

  1. Bullock TL et al. (1994), Biochemistry, 33, 11127-11134. Structure of the Complex of L-Benzylsuccinate with Wheat Serine Carboxypeptidase II at 2.0-.ANG. Resolution. DOI:10.1021/bi00203a009. PMID:7727364.
  2. Mortensen UH et al. (1994), Biochemistry, 33, 508-517. Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate. DOI:10.1021/bi00168a016. PMID:7904479.
  3. Stennicke HR et al. (1994), Protein Eng, 7, 911-916. Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y. DOI:10.1093/protein/7.7.911. PMID:7971953.
  4. Liao DI et al. (1992), Biochemistry, 31, 9796-9812. Refined atomic model of wheat serine carboxypeptidase II at 2.2-.ANG. resolution. DOI:10.1021/bi00155a037. PMID:1390755.

Step 1. His397, of the Ser146-His397-Asp338 triad, deprotonates Ser146.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp338(75)B hydrogen bond acceptor, increase basicity
Ser146(153)A hydrogen bond donor
His397(127)B hydrogen bond donor, hydrogen bond acceptor, proton acceptor
Ser146(153)A proton donor

Chemical Components

proton transfer

Step 2. Ser146 attacks the carbonyl carbon of the substrate in a nucleophilic addition, resulting in the formation of a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly53(57)A (main-N) electrostatic stabiliser, hydrogen bond donor
Asp338(75)B hydrogen bond acceptor, electrostatic stabiliser
Ser146(153)A hydrogen bond acceptor
His397(127)B hydrogen bond donor, electrostatic stabiliser
Tyr147(154)A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser146(153)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 3. The tetrahedral intermediate collapses, cleaving the C-N bond, the nitrogen of which deprotonates His397.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly53(57)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp338(75)B hydrogen bond acceptor, increase acidity
Ser146(153)A covalently attached, hydrogen bond acceptor
His397(127)B hydrogen bond donor
Tyr147(154)A (main-N) hydrogen bond donor, electrostatic stabiliser
His397(127)B proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation

Step 4. His397 deprotonates water, which initiates a nucleophilic attack upon the carbonyl carbon of the acyl-enzyme substrate, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly53(57)A (main-N) electrostatic stabiliser, hydrogen bond donor
Asp338(75)B hydrogen bond acceptor, increase basicity
Ser146(153)A covalently attached, hydrogen bond acceptor
His397(127)B hydrogen bond acceptor, hydrogen bond donor
Tyr147(154)A (main-N) hydrogen bond donor, electrostatic stabiliser
His397(127)B proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 5. The tetrahedral intermediate collapses, eliminating Ser146, which deprotonates His397.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly53(57)A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp338(75)B hydrogen bond acceptor, increase acidity
Ser146(153)A hydrogen bond acceptor
His397(127)B hydrogen bond donor
Tyr147(154)A (main-N) hydrogen bond donor, electrostatic stabiliser
His397(127)B proton donor
Ser146(153)A proton acceptor, nucleofuge

Chemical Components

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

Step 1. His397, of the Ser146-His397-Asp338 triad, deprotonates Ser146.

Step 2. Ser146 attacks the carbonyl carbon of the substrate in a nucleophilic addition, resulting in the formation of a tetrahedral intermediate.

Step 3. The tetrahedral intermediate collapses, cleaving the C-N bond, the nitrogen of which deprotonates His397.

Step 4. His397 deprotonates water, which initiates a nucleophilic attack upon the carbonyl carbon of the acyl-enzyme substrate, forming a tetrahedral intermediate.

Step 5. The tetrahedral intermediate collapses, eliminating Ser146, which deprotonates His397.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Alex Gutteridge, Craig Porter, Katherine Ferris