Subtilisin

 

Subtilisin is an extracellular enzyme secreted by Bacillus amyloliquefaciens and belongs to the alpha/beta subtilase family. It is a serine-class endoprotease, hydrolysing peptide bonds with broad specificity and a preference for a large uncharged residue in the P1 binding site.

 

Reference Protein and Structure

Sequence
P00782 UniProt (3.4.21.62) IPR015500 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus amyloliquefaciens (Bacteria) Uniprot
PDB
1s01 - LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING (1.7 Å) PDBe PDBsum 1s01
Catalytic CATH Domains
3.40.50.200 CATHdb (see all for 1s01)
Click To Show Structure

Enzyme Reaction (EC:3.4.21.62)

dipeptide zwitterion
CHEBI:90799ChEBI
+
water
CHEBI:15377ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Bacillus subtilis alkaline proteinase, Bacillus subtilis alkaline proteinase bioprase, ALK-enzyme, SP 266, Alcalase, Alcalase 0.6L, Alcalase 2.5L, Bacillopeptidase A, Bacillopeptidase B, Bioprase AL 15, Bioprase APL 30, Colistinase, Esperase, Genenase I, Kazusase, Maxatase, Opticlean, Orientase 10B, Protease S, Protease VIII, Protease XXVII, Protin A 3L, Savinase, Savinase 16.0L, Savinase 32.0 L EX, Savinase 4.0T, Savinase 8.0L, Subtilisin BL, Subtilisin DY, Subtilisin E, Subtilisin GX, Subtilisin J, Subtilisin S41, Subtilisin Sendai, Subtilopeptidase, Superase, Thermoase, Thermoase PC 10,

Enzyme Mechanism

Introduction

His 171 acts as a general base, deprotonating Ser 328. Ser 328 performs nucleophilic attack on the carbonyl carbon of the amide bond. This results in a tetrahedral transition state, which is stabilised through Coulombic interactions with protonated His 171, hydrogen bonding with the backbone amide of Ser 328 and the amide side-chain of Asn 262. The transition state is also stabilised by hydrogen bonding between the P1 amide nitrogen and the carbonyl oxygen of Ser 232. His 171 is stabilised by electrostatic interactions with Asp 139. The tetrahedral transition state collapses, forming an acyl-enzyme and His 171 acts as a general acid, protonating the amide leaving group. His 171 acts as a general base, deprotonating a water molecule. The activated water molecule performs nucleophilic attack upon the acyl enzyme, forming a tetrahedral transition state. The tetrahedral transition state collapses, forming the acid component of the substrate and Ser 328. His 171 acts as a general acid, protonating the leaving group Ser 328.

Catalytic Residues Roles

UniProt PDB* (1s01)
Ser328 (main-N) Ser221A (main-N) The backbone amide group of Ser 328 hydrogen bonds with the tetrahedral transition state, stabilising it. electrostatic interaction, electrostatic stabiliser
Ser328 Ser221A Ser 328 is deprotonated by His 171 and performs nucleophilic attack on the carbonyl carbon of the amide bond, resulting in a tetrahedral transition state. nucleofuge, nucleophile, proton acceptor, proton donor
Asn262 Asn155A The amide side-chain of Asn 262 stabilises the first tetrahedral transition state through hydrogen bonding. electrostatic interaction, electrostatic stabiliser
Asp139 Asp32A Asp 139 stabilises the positive charge on His 171 through electrostatic interactions. electrostatic interaction, electrostatic stabiliser
His171 His64A 1: His 171 acts as a general base, deprotonating Ser 328. 2: Protonated His 171 stabilises the tetrahedral transition state through Coulombic interactions. 3: Protonated His 171 acts as a general acid, protonating the amide leaving group. 4: His 171 acts as a general base, deprotonating a water molecule. 5: Protonated His 171 acts as a general acid, protonating the leaving group Ser 328. proton acceptor, proton donor
*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, intermediate formation, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Bryan P et al. (1986), Proc Natl Acad Sci U S A, 83, 3743-3745. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin. DOI:10.1073/pnas.83.11.3743. PMID:3520553.
  2. Plou FJ et al. (1996), J Mol Biol, 257, 1088-1111. Characterization of the Electrostatic Perturbation of a Catalytic Site (Cys)-S–/(His)-Im+H Ion-pair in One Type of Serine Proteinase Architecture by Kinetic and Computational Studies on Chemically Mutated Subtilisin Variants. DOI:10.1006/jmbi.1996.0225.
  3. Takeuchi Y et al. (1991), J Mol Biol, 221, 309-325. Refined crystal structure of the complex of subtilisin BPN? and Streptomyces subtilisin inhibitor at 1.8 p resolution. DOI:10.1016/0022-2836(91)90821-m. PMID:1920411.
  4. Carter P et al. (1990), Proteins, 7, 335-342. Functional interaction among catalytic residues in subtilisin BPN′. DOI:10.1002/prot.340070405. PMID:2199971.
  5. Blow DM et al. (1969), Nature, 221, 337-340. Role of a Buried Acid Group in the Mechanism of Action of Chymotrypsin. DOI:10.1038/221337a0. PMID:5764436.

Step 1. His171 deprotonates Ser328 activating it to attack the carbon of the peptide bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn155A electrostatic stabiliser
Asp32A electrostatic stabiliser
Ser221A (main-N) electrostatic stabiliser
Ser221A nucleophile, proton donor
His64A proton acceptor

Chemical Components

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

Step 2. The tetrahedral transition state collapses, forming an acyl-enzyme and His171 acts as a general acid, protonating the amide leaving group

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp32A electrostatic stabiliser
Asn155A electrostatic stabiliser
Ser221A (main-N) electrostatic stabiliser
His64A proton donor

Chemical Components

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

Step 3. His171 abstracts a proton from a water which in turn activates it to attack the carbon of the ester bond in a nucleophilic addition to produce another transition state tetrahedral intermediate

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp32A electrostatic interaction
Asn155A electrostatic interaction
Ser221A (main-N) electrostatic interaction
His64A proton acceptor

Chemical Components

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

Step 4. The tetrahedral transition state collapses, forming the acid component of the substrate and Ser328. His171 acts as a general acid, protonating the leaving group Ser328 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp32A electrostatic stabiliser
Asn155A electrostatic stabiliser
Ser221A (main-N) electrostatic stabiliser
Ser221A proton acceptor
His64A proton donor
Ser221A nucleofuge

Chemical Components

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

Step 1. His171 deprotonates Ser328 activating it to attack the carbon of the peptide bond in a nucleophilic addition.

Step 2. The tetrahedral transition state collapses, forming an acyl-enzyme and His171 acts as a general acid, protonating the amide leaving group

Step 3. His171 abstracts a proton from a water which in turn activates it to attack the carbon of the ester bond in a nucleophilic addition to produce another transition state tetrahedral intermediate

Step 4. The tetrahedral transition state collapses, forming the acid component of the substrate and Ser328. His171 acts as a general acid, protonating the leaving group Ser328 which returns the enzyme to its native state.

Products.

Contributors

Fiona J. E. Morgan, Gemma L. Holliday, Christian Drew, Craig Porter, Charity Hornby