Alpha-lytic endopeptidase

 

Alpha-lytic protease is a bacterial homologue of the chymotrypsin family of serine proteases. The role of this unusually stable extracellular enzyme is to lyse microorganisms and proteolyse their contents to provide nutrients for the host bacterium. The enzyme preferentially cleaves Ala-|-Xaa, Val-|-Xaa in bacterial cell walls, elastin and other proteins. Belongs to the peptidase S1E family.

 

Reference Protein and Structure

Sequence
P00778 UniProt (3.4.21.12) IPR001316 (Sequence Homologues) (PDB Homologues)
Biological species
Lysobacter enzymogenes (Bacteria) Uniprot
PDB
1ssx - 0.83A resolution crystal structure of alpha-lytic protease at pH 8 (0.83 Å) PDBe PDBsum 1ssx
Catalytic CATH Domains
2.40.10.10 CATHdb (see all for 1ssx)
Click To Show Structure

Enzyme Reaction (EC:3.4.21.12)

Ala-Ser
CHEBI:73394ChEBI
+
water
CHEBI:15377ChEBI
→
L-alanine
CHEBI:16977ChEBI
+
L-serine
CHEBI:17115ChEBI
Alternative enzyme names: Alpha-lytic protease, Alpha-lytic proteinase, Mycobacterium sorangium alpha-lytic proteinase, Myxobacter 495 alpha-lytic proteinase, Myxobacter alpha-lytic proteinase,

Enzyme Mechanism

Introduction

Alpha-lytic protease employs a classic Ser-His-Asp triad. His 235 removes a proton from Ser 342 as the latter nucleophilically attacks the peptide carbonyl to give a tetrahedral intermediate which is stabilised by the NH groups of Gly 340 and Ser 342. Breakdown of the intermediate with protonation of the departing amine by His 235 generates a covalent acyl-enzyme intermediate. This is then hydrolysed using a water molecule that is deprotonated by His 235. Asp 262 and Ser358 functions to modify the pKa of His 235, enabling it to deprotonate Ser 342 and water.

Catalytic Residues Roles

UniProt PDB* (1ssx)
Gly340 (main-N), Ser342 (main-N) Gly193(141)A (main-N), Ser195(143)A (main-N) Forms part of the oxyanion hole that stabilises the tetrahedral intermediate. electrostatic stabiliser
Ser342 Ser195(143)A Acts as a nucleophile to attack the peptide bond carbonyl. Its backbone NH forms part of the oxyanion hole which stabilises the tetrahedral intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
His235 His57(36)A Removes proton from Ser 195 as the Ser 195 acts as a nucleophile to attack the peptide carbonyl. Acts as a general acid to protonate the departing amine leaving group. Later deprotonates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. proton acceptor, proton donor
Ser358 (main-C) Ser214(159)A (main-C) Backbone carbonyl forms a weak C-H...O hydrogen bond to C-epsilon1 of His 57. This is proposed to help delocalise the charge of the histidine ring, weakening the epsilon2N-H bond and promoting breakdown of the tetrahedral intermediate to the acyl enzyme. electrostatic stabiliser
Asp262 Asp102(63)A The Asp 102 carboxylate modifies the pKa of His 57 via stabilising it in its protonated state. 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, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Fuhrmann CN et al. (2004), J Mol Biol, 338, 999-1013. The 0.83Å Resolution Crystal Structure of α-Lytic Protease Reveals the Detailed Structure of the Active Site and Identifies a Source of Conformational Strain. DOI:10.1016/j.jmb.2004.03.018. PMID:15111063.
  2. Wahlgren WY et al. (2011), J Biol Chem, 286, 3587-3596. The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action. DOI:10.1074/jbc.M110.161604. PMID:21097875.
  3. Hedstrom L (2002), Chem Rev, 34, 4501-4524. Serine Protease Mechanism and Specificity. DOI:10.1002/chin.200306269. PMID:12475199.
  4. Sauter NK et al. (1998), Nat Struct Biol, 5, 945-950. Structure of α-lytic protease complexed with its pro region. DOI:10.1038/2919. PMID:9808037.
  5. Bone R et al. (1987), Biochemistry, 26, 7609-7614. Serine protease mechanism: structure of an inhibitory complex of .alpha.-lytic protease and a tightly bound peptide boronic acid. DOI:10.1021/bi00398a012. PMID:3122831.

Step 1. His235 deprotonates Ser342 activating it so it can then go on to attack the carbon of the peptide bond producing the oxyanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(63)A electrostatic stabiliser
Gly193(141)A (main-N) electrostatic stabiliser
Ser214(159)A (main-C) electrostatic stabiliser
Ser195(143)A (main-N) electrostatic stabiliser
His57(36)A proton acceptor
Ser195(143)A proton donor, nucleophile

Chemical Components

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

Step 2. The tetrahedral intermediate collapses with expulsion of the leaving group, assisted by protonated His 235 acting as a general acid which protonates the N-terminal product, to yield the acylenzyme intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(63)A electrostatic stabiliser
Gly193(141)A (main-N) electrostatic stabiliser
Ser195(143)A (main-N) electrostatic stabiliser
Ser214(159)A (main-C) electrostatic stabiliser
His57(36)A proton donor

Chemical Components

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

Step 3. His235 abstracts a proton from a water which activates it to attack the carbon of the acylenzyme, forming a second tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(63)A electrostatic stabiliser
Gly193(141)A (main-N) electrostatic stabiliser
Ser195(143)A (main-N) electrostatic stabiliser
Ser214(159)A (main-C) electrostatic stabiliser
His57(36)A proton acceptor

Chemical Components

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

Step 4. This intermediate collapses, expelling Ser 342 as the leaving group, assisted by the protonated His 235 acting as a general acid, and forming the carboxylic acid product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(63)A electrostatic stabiliser
Gly193(141)A (main-N) electrostatic stabiliser
Ser195(143)A (main-N) electrostatic stabiliser
Ser214(159)A (main-C) electrostatic stabiliser
Ser195(143)A proton acceptor, nucleofuge
His57(36)A proton donor

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. His235 deprotonates Ser342 activating it so it can then go on to attack the carbon of the peptide bond producing the oxyanion intermediate.

Step 2. The tetrahedral intermediate collapses with expulsion of the leaving group, assisted by protonated His 235 acting as a general acid which protonates the N-terminal product, to yield the acylenzyme intermediate.

Step 3. His235 abstracts a proton from a water which activates it to attack the carbon of the acylenzyme, forming a second tetrahedral intermediate.

Step 4. This intermediate collapses, expelling Ser 342 as the leaving group, assisted by the protonated His 235 acting as a general acid, and forming the carboxylic acid product.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby