Signal peptidase I

 

Escherichia coli type I signal peptidase is a membrane-bound serine endopeptidase that cleaves the amino-terminal signal sequence from secretory proteins and some membrane proteins. Evolutionarily the enzyme belongs to the protease clan SF and the protease family S26.

 

Reference Protein and Structure

Sequence
P00803 UniProt (3.4.21.89) IPR000223 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1t7d - Crystal structure of Escherichia coli type I signal peptidase in complex with a lipopeptide inhibitor (2.47 Å) PDBe PDBsum 1t7d
Catalytic CATH Domains
2.10.109.10 CATHdb (see all for 1t7d)
Click To Show Structure

Enzyme Reaction (EC:3.4.21.89)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: Escherichia coli leader peptidase, HOSP, PuIO prepilin peptidase, SPC, Bacterial leader peptidase 1, Eukaryotic signal peptidase, Eukaryotic signal proteinase, Leader peptidase, Leader peptidase I, Leader peptide hydrolase, Leader proteinase, Pilin leader peptidase, Prokaryotic leader peptidase, Prokaryotic signal peptidase, Prokaryotic signal proteinase, Propeptidase, Signal peptidase, Signal peptide hydrolase, Signal peptide peptidase, Signal proteinase, Signalase, SPase I, Phage-procoat-leader peptidase, Bacterial leader peptidase I,

Enzyme Mechanism

Introduction

Signal peptidase uses a Ser-Lys dyad to catalyse attack on the si-face of the peptide bond. Ser 91 acts as a nucleophile to attack the carbonyl group while Lys 146 deprotonates the attacking Ser 91. The resulting tetrahedral intermediate is stabilised by an oxyanion hole consisting of the backbone NH of Ser 91 and the side chain hydroxyl of Ser 89. Collapse of the tetrahedral intermediate with protonation of the departing amine by Lys 146 generates an acyl-enzyme intermediate; this is then hydrolysed by a water molecule that is deprotonated by Lys 146. Lys 146 is located in a hydrophobic environment in the protein; this reduces its pKa and so allows it to carry out its role as an acid/base.

Catalytic Residues Roles

UniProt PDB* (1t7d)
Ser279 Ser278(205)B Stabilises the Lys residue. electrostatic stabiliser
Ser89 Ser88(15)B Side chain hydroxyl forms part of the oxyanion hole that stabilises the tetrahedral intermediate. electrostatic stabiliser
Ser91 (main-N), Ser91 Ser90(17)B (main-N), Ser90(17)B Attacks the peptide bond to form an acyl enzyme intermediate. Backbone NH forms part of the oxyanion hole that stabilises the tetrahedral intermediate. electrostatic stabiliser
Lys146 Lys145(72)B Deprotonates Ser 91 as Ser 91 attacks the peptide carbonyl. Protonates the departing amine group during collapse of the tetrahedral intermediate. Deprotonates a water molecule during hydrolysis of the acyl-enzyme intermediate. 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, 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. Paetzel M et al. (2004), J Biol Chem, 279, 30781-30790. Crystallographic and Biophysical Analysis of a Bacterial Signal Peptidase in Complex with a Lipopeptide-based Inhibitor. DOI:10.1074/jbc.m401686200. PMID:15136583.
  2. Paetzel M (2014), Biochim Biophys Acta, 1843, 1497-1508. Structure and mechanism of Escherichia coli type I signal peptidase. DOI:10.1016/j.bbamcr.2013.12.003. PMID:24333859.
  3. Carlos JL et al. (2000), Biochemistry, 39, 7276-7283. Mutational Evidence of Transition State Stabilization by Serine 88 inEscherichia coliType I Signal Peptidase†,‡. DOI:10.1021/bi000301l. PMID:10852727.
  4. Paetzel M et al. (1998), Nature, 396, 186-190. Crystal structure of a bacterial signal peptidase in complex with a beta-lactam inhibitor. DOI:10.1038/24196. PMID:9823901.
  5. Tschantz WR et al. (1993), J Biol Chem, 268, 27349-27354. A serine and a lysine residue implicated in the catalytic mechanism of the Escherichia coli leader peptidase. PMID:8262975.
  6. Sung M et al. (1992), J Biol Chem, 267, 13154-13159. Identification of potential active-site residues in the Escherichia coli leader peptidase. PMID:1618816.

Step 1. Lys 146 deprotonates Ser 91 which activates it to nucleophilically attack the carbon of the carbonyl to form the oxyanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser88(15)B electrostatic stabiliser
Ser90(17)B (main-N) electrostatic stabiliser
Ser278(205)B electrostatic stabiliser
Ser90(17)B proton donor
Lys145(72)B proton acceptor
Ser90(17)B nucleophile

Chemical Components

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

Step 2. Lys 146 protonates the amine of the peptide bond which results in the collapse of the tetrahedral intermediate and the cleavage of the peptide bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser88(15)B electrostatic stabiliser
Ser90(17)B (main-N) electrostatic stabiliser
Ser278(205)B electrostatic stabiliser
Lys145(72)B proton donor

Chemical Components

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

Step 3. Lys 146 abstracts a proton from a water which activates it to attack the carbon of the ester bond in a nucleophilic addition once again producing the oxyanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser88(15)B electrostatic stabiliser
Ser90(17)B (main-N) electrostatic stabiliser
Ser278(205)B electrostatic stabiliser
Lys145(72)B proton acceptor

Chemical Components

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

Step 4. The tetrahedral intermediate collapse again resulting in the release of Ser91 which then accepts a proton from Lys 146 returning the active site to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser88(15)B electrostatic stabiliser
Ser90(17)B (main-N) electrostatic stabiliser
Ser278(205)B electrostatic stabiliser
Lys145(72)B proton donor
Ser90(17)B nucleofuge, proton acceptor

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. Lys 146 deprotonates Ser 91 which activates it to nucleophilically attack the carbon of the carbonyl to form the oxyanion intermediate.

Step 2. Lys 146 protonates the amine of the peptide bond which results in the collapse of the tetrahedral intermediate and the cleavage of the peptide bond.

Step 3. Lys 146 abstracts a proton from a water which activates it to attack the carbon of the ester bond in a nucleophilic addition once again producing the oxyanion intermediate.

Step 4. The tetrahedral intermediate collapse again resulting in the release of Ser91 which then accepts a proton from Lys 146 returning the active site to its native state.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby