Caspase-9

 

Caspase-9 from Homo sapiens is a cysteine dependent aspartate specific protease. When activated, it will cleave the Caspase-3 peptide bond between Asp 175 and Ser 176. On existence as a monomer, Caspase-9 is inactive, but on dimerizing which is a result of the release of cytochrome c from the mitochondria cleaving procaspase-9 it becomes active, or it can be activated by making a complex with APAF1. It is involved in the process of apoptosis (programmed cell death) which is a central role in the development and homeostasis of an organism and Caspase-9 is linked to the normal development of the central nervous system.

 

Reference Protein and Structure

Sequences
P98170 UniProt (2.3.2.27)
P55211 UniProt (3.4.22.62) IPR033171 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1nw9 - STRUCTURE OF CASPASE-9 IN AN INHIBITORY COMPLEX WITH XIAP-BIR3 (2.4 Å) PDBe PDBsum 1nw9
Catalytic CATH Domains
3.40.50.1460 CATHdb (see all for 1nw9)
Click To Show Structure

Enzyme Reaction (EC:3.4.22.62)

water
CHEBI:15377ChEBI
+
dipeptide
CHEBI:46761ChEBI
→
L-alpha-amino acid
CHEBI:15705ChEBI
Alternative enzyme names: CASP-9, ICE-like apoptotic protease 6, ICE-LAP6, Apoptotic protease Mch-6, Apoptotic protease-activating factor 3, APAF-3,

Enzyme Mechanism

Introduction

The backbone NH's of Cys 287 and Gly 238 form the oxyanion hole, and activate the scissile carbonyl bond for nucleophilic attack. His 237 is made more basic through hydrogen bonding with the oxygen atom from the backbone of Arg 178. His 237 deprotonates Cys 285 side chain, activating it so the S atom nucleophilically attacks the carbonyl, forming a negatively charged, tetrahedral intermediate. The intermediate is stabilised by hydrogen bonding to the residues forming the oxyanion hole. The carbonyl is reformed, and the leaving group amine is protonated by the previously protonated His 237. His 237 then activates a water molecule for nucleophilic attack on the carbonyl by deprotonating it. The negatively charged, tetrahedral intermediate is again stabilised by the oxyanion hole. The carbonyl is again reformed, and the protonated His 237 donates a proton to the leaving group Cys 285.

Catalytic Residues Roles

UniProt PDB* (1nw9)
Cys287 (main-N), Gly238 (main-N) Cys287(148)B (main-N), Gly238(99)B (main-N) The backbone NH of Gly 238, along with that of Cys 285, forms the oxyanion hole. Hydrogen bonds between the residue and the carbonyl oxygen of the scissile bond help to activate the carbonyl, and also to stabilise the negatively charged, tetrahedral intermediate. electrostatic stabiliser
His237 His237(98)B His 237 deprotonates Cys 287, which then goes on to nucleophilically attack the substrate. The protonated form of His 237 then donates a proton to the leaving group amine as the carbonyl is reformed. His 237 then deprotonates a water molecule which goes on to nucleophilically attack the carbonyl. Protonated His 237 then donates a proton back to the leaving group Cys 287 as the carbonyl is once more reformed. proton acceptor, proton donor
Cys287 Cys287(148)B The backbone NH of Cys 287 helps form the oxyanion hole by donating a H-bond to the carbonyl oxygen of the scissile bond. This makes the carbonyl more susceptible to nucleophilic attack, and also stabilises the negatively charged, tetrahedral intermediate. The side chain of Cys 287 is deprotonated by His 237, and nucleophilically attacks the carbonyl of the scissile bond, forming a negatively charged, tetrahedral intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
Arg178 (main-C) Arg178(39)B (main-C) The carbonyl oxygen of Arg 178 acts electrostatically on His 237, making it more basic, and thereby making it more ready to accept a proton from Cys 287. 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, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Chou KC et al. (2000), FEBS Lett, 470, 249-256. Prediction of the tertiary structure of a caspase-9/inhibitor complex. DOI:10.1016/s0014-5793(00)01333-8. PMID:10745077.
  2. Stennicke HR et al. (1999), Cell Death Differ, 6, 1054-1059. Catalytic properties of the caspases. DOI:10.1038/sj.cdd.4400599. PMID:10578173.

Step 1. His237 deprotonates Cys287, activating it to so it can nucleophilically attack the carbon of the carbonyl which produces the oxyanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly238(99)B (main-N) electrostatic stabiliser
Arg178(39)B (main-C) electrostatic stabiliser
Cys287(148)B (main-N) electrostatic stabiliser
His237(98)B proton acceptor
Cys287(148)B nucleophile, proton donor

Chemical Components

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

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. His237 then donates a proton to the amino group of the N-terminal product preventing reformation of the peptide bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg178(39)B (main-C) electrostatic stabiliser
Gly238(99)B (main-N) electrostatic stabiliser
Cys287(148)B (main-N) electrostatic stabiliser
His237(98)B proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed

Step 3. His237 deprotonates a water, activating it so that it can attack the carbon of the thioester bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg178(39)B (main-C) electrostatic stabiliser
Gly238(99)B (main-N) electrostatic stabiliser
Cys287(148)B (main-N) electrostatic stabiliser
His237(98)B proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates an elimination which results in the cleavage of the thioester bond. Cys287 now accepts a proton from His237 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg178(39)B (main-C) electrostatic stabiliser
Gly238(99)B (main-N) electrostatic stabiliser
Cys287(148)B (main-N) electrostatic stabiliser
His237(98)B proton donor
Cys287(148)B proton acceptor, nucleofuge

Chemical Components

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

Step 1. His237 deprotonates Cys287, activating it to so it can nucleophilically attack the carbon of the carbonyl which produces the oxyanion intermediate.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. His237 then donates a proton to the amino group of the N-terminal product preventing reformation of the peptide bond.

Step 3. His237 deprotonates a water, activating it so that it can attack the carbon of the thioester bond in a nucleophilic addition.

Step 4. The oxyanion initiates an elimination which results in the cleavage of the thioester bond. Cys287 now accepts a proton from His237 which returns the enzyme to its native state.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, Charity Hornby