Staphopain

 

Staphopain B is a possible virulence factor from Staphylococcus aureus, and is a member of the papain superfamily of cysteine proteases. It is able to degrade elastin, fibrogen, fibronectin and kininogen. Exhibits a strong preference for substrates where arginine is preceded by a hydrophobic amino acid. Promotes detachment of primary human keratinocytes. Along with other extracellular proteases is involved in colonization and infection of human tissues

Staphostatin B is a specific inhibitor of staphopain B encoded in the same operon. Inhibition of staphopains is a target for treatment of antibiotic-resistant S. aureus infections. It is a produced as an inactive proenzyme, which is comprised of the active part of the enzyme, the mature form, and a covalently bonded inhibitory pro-region. Activation of the enzyme requires cleavage of the N terminal inhibitory pro-region (staphostatin B)

 

Reference Protein and Structure

Sequence
P0C1S6 UniProt (3.4.22.-) IPR008750 (Sequence Homologues) (PDB Homologues)
Biological species
Staphylococcus aureus (Bacteria) Uniprot
PDB
1x9y - The prostaphopain B structure (2.5 Å) PDBe PDBsum 1x9y
Catalytic CATH Domains
3.90.70.10 CATHdb (see all for 1x9y)
Click To Show Structure

Enzyme Reaction (EC:3.4.22.28)

water
CHEBI:15377ChEBI
+
dipeptide
CHEBI:46761ChEBI
→
L-alpha-amino acid
CHEBI:15705ChEBI
Alternative enzyme names: 3C protease, 3C proteinase, Coxsackievirus 3C proteinase, Cysteine proteinase 3C, Foot-and-mouth protease 3C, Foot-and-mouth-disease virus proteinase 3C, Hepatitis A virus 3C proteinase, Picornavirus endopeptidase 3C, Poliovirus protease 3C, Poliovirus proteinase 3C, Protease 3C, Rhinovirus protease 3C, Rhinovirus proteinase 3C, Tomato ringspot nepovirus 3C-related protease,

Enzyme Mechanism

Introduction

Cys 243 and His 340 are held as a thiolate-imidazolium ion pair. The thiolate is nucleophilic and attacks the electrophilic scissile amide bond of the substrate to form a tetrahedral intermediate. This intermediate collapses, with His 340 acting as a general acid to the leaving group, and results in acylation of Cys 243.
Deacylation is by hydrolysis, with His 340 acting as a general base to activate the water molecule. Water is a nucleophile and attacks the electrophilic thioester group of the Cys 243-substrate bond, forming a tetrahedral intermediate. This intermediate collapses, with the cationic His 340 stabilising the Cys 243 thiolate leaving group, and the formation of the product.

Catalytic Residues Roles

UniProt PDB* (1x9y)
Cys243 (main-N), Gln237 Cys243(209)A (main-N), Gln237(203)A Gln 237 acts as an oxyanion hole and acts by stabilising the negative charge of the transition states and tetrahedral intermediates via its side chain amide. By homology with other papain-type enzymes, the main chain amide of Cys 243 is assumed to also form part of the oxyanion hole. electrostatic stabiliser
Cys243 Cys243(209)A Cys 243 is held as a thiolate and is the nucleophile that attacks the scissile amide electrophile, forming a tetrahedral intermediate. Collapse of this intermediate results in acylation of Cys 243. After hydrolysis of the acyl-enzyme, Cys 243 is the leaving group.The thiolate ion is stabilised by the formation of an active site ion pair with the His 340 imidazole ring. The main chain NH of Cys 243 forms part of the oxyanion hole, which stabilises the transition state. nucleofuge, nucleophile, proton acceptor, proton donor
His340 His340(306)A The basic side chain of the His residue deprotonates the cysteine thiol to activate it towards nucleophilic attack of the substrate peptide bond. The imidazole ring of His 340 forms an ion pair with Cys 243. His 340 forms a hydrogen bond to the Asn 360 side chain amide oxygen to stabilise the ion pair and keep the imidazole ring in favourable orientation. proton acceptor, proton donor
Asn360 Asn360(326)A Asn 360 is adjacent to the catalytic His 340, and its side chain amide oxygen is hydrogen bonded to the N(e2)H of His 340. This effect of this is to stabilise the cationic form of His, increase the basicity of the neutral form of His, and also keep the imidazole ring of the His residue in favourable orientation. 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

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

References

  1. Filipek R et al. (2004), Biochemistry, 43, 14306-14315. Prostaphopain B Structure:  A Comparison of Proregion-Mediated and Staphostatin-Mediated Protease Inhibition†,‡. DOI:10.1021/bi048661m. PMID:15518582.
  2. Filipek R et al. (2005), J Biol Chem, 280, 14669-14674. A Comparison of Staphostatin B with Standard Mechanism Serine Protease Inhibitors. DOI:10.1074/jbc.m411792200. PMID:15644332.
  3. Filipek R et al. (2003), J Biol Chem, 278, 40959-40966. The Staphostatin-Staphopain Complex: A FORWARD BINDING INHIBITOR IN COMPLEX WITH ITS TARGET CYSTEINE PROTEASE. DOI:10.1074/jbc.m302926200. PMID:12874290.
  4. Ballistreri A et al. (2001), Int J Biol Macromol, 29, 107-114. Biosynthesis and structural characterization of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas aeruginosa from fatty acids. DOI:10.1016/s0141-8130(01)00154-4. PMID:11518582.
  5. Harrison MJ et al. (1997), J Am Chem Soc, 119, 12285-12291. Catalytic Mechanism of the Enzyme Papain:  Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. DOI:10.1021/ja9711472.

Step 1. His340 deprotonates Cys243 which nucleophilically attack the carbonyl carbon.

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Catalytic Residues Roles

Residue Roles
Gln237(203)A electrostatic stabiliser
Cys243(209)A (main-N) electrostatic stabiliser
Asn360(326)A modifies pKa, electrostatic stabiliser
Cys243(209)A nucleophile
His340(306)A proton acceptor
Cys243(209)A proton donor

Chemical Components

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

Step 2. His340 transfer a proton to the amide nitrogen which initiates an elimination from the oxyanion which results in the cleavage of the peptide bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln237(203)A electrostatic stabiliser
Cys243(209)A (main-N) electrostatic stabiliser
Asn360(326)A electrostatic stabiliser, modifies pKa
His340(306)A proton donor

Chemical Components

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

Step 3. His340 abstracts a proton from a water which can nucleophilically attack the carbonyl carbon.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln237(203)A electrostatic stabiliser
Cys243(209)A (main-N) electrostatic stabiliser
Asn360(326)A electrostatic stabiliser, modifies pKa
His340(306)A 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 acyl-enzyme intermediate and the release of Cys243 means it can then accept a proton from His340.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln237(203)A electrostatic stabiliser
Cys243(209)A (main-N) electrostatic stabiliser
Asn360(326)A electrostatic stabiliser, modifies pKa
Cys243(209)A nucleofuge, proton acceptor
His340(306)A proton donor

Chemical Components

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

Step 1. His340 deprotonates Cys243 which nucleophilically attack the carbonyl carbon.

Step 2. His340 transfer a proton to the amide nitrogen which initiates an elimination from the oxyanion which results in the cleavage of the peptide bond.

Step 3. His340 abstracts a proton from a water which can nucleophilically attack the carbonyl carbon.

Step 4. The oxyanion initiates an elimination which results in the cleavage of the acyl-enzyme intermediate and the release of Cys243 means it can then accept a proton from His340.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Charity Hornby