HslU---HslV peptidase

 

HslVU is an ATP-dependent prokaryotic proteasome. It is a homolog of the eukaryotic 26S proteasome. It is responsible for the degradation of a majority of proteins in the cell, including regulatory protein factors and abnormally folded proteins. HslVU has two protein components, HslV and HslU. HslV is a protease. By itself, HslV has a low catalytic activity. Its activity is enhanced greatly by the interaction with HslU to form HslVU complex. HslU consists of ATPase and chaperone activities. It belongs to the Hsp100/Clp family of molecular chaperones, which is a member of the extended AAA(ATPase associated with a variety of cellular activities) family. It was found that the degradation of polypeptides by HslVU has an ATP requirement. For at least one substrate, SulA protein, ATP hydrolysis is required for degradation. Though ATP hydrolysis does not seem to be mandatory.

 

Reference Protein and Structure

Sequences
P0A7B8 UniProt (3.4.25.2)
P0A6H5 UniProt IPR022281 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ht1 - Nucleotide-Dependent Conformational Changes in a Protease-Associated ATPase HslU (2.8 Å) PDBe PDBsum 1ht1
Catalytic CATH Domains
3.60.20.10 CATHdb (see all for 1ht1)
Click To Show Structure

Enzyme Reaction (EC:3.4.25.2)

water
CHEBI:15377ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: ClpQ, ClpYQ, ClpYQ protease, HslUV, HslV-HslU, HslV peptidase, ATP-dependent HslV-HslU proteinase, Caseinolytic protease X, Caseinolytic proteinase X, ClpXP ATP-dependent protease, ClpXP protease, ClpXP serine proteinase, Escherichia coli ClpXP serine proteinase, HslUV protease, HslUV proteinase, HslVU protease, HslVU proteinase, Protease ClpYQ, Protease CodWX, Protease HslVU, Proteinase ClpYQ, Proteinase HslUV,

Enzyme Mechanism

Introduction

HslV is an N-terminal nucleophilic (Ntn) hydrolase. N-terminal threonine residue(Thr1) hydroxyl group is deprotonated by its own -NH2 group as it attacks the substrate carbonyl to form a tetrahedral intermediate, which is stabilised by an oxyanion hole formed by the backbone NH group of Gly 45. Collapse of the tetrahedral intermediate with protonation of the departing amine group by the terminal -NH3+ group generates an acyl enzyme intermediate; this is then attacked by a water molecule that is deprotonated by the terminal -NH2 group of Thr 1 acting again as a general base. The nearby Lys 33 (in its protonated form) hydrogen bonds with the hydroxyl group oxygen atom of Thr 1 and provide a positive charge to promote deprotonation of this group in the first step of the reaction. Conserved Ser124 appears to hydrogen bond with the Thr1 N and maybe necessary to control the protonation of Thr1 N. In addition, the binding and hydrolysis of ATP by HsIU is essential for activation of protease activity in HsIV.

Catalytic Residues Roles

UniProt PDB* (1ht1)
Thr2, Thr2 (N-term) Thr1A(E), Thr1A(E) (N-term) Its hydroxyl group, upon deprotonation by its -NH2, acts as a nucleophile to attack the substrate carbonyl to form a tetrahedral intermediate. Its NH3+ group then protonates the departing amine group to generate the acyl enzyme intermediate; this is then attacked by a water molecule that is deprotonated by the terminal -NH2 group of Thr 1. nucleofuge, nucleophile, proton acceptor, proton donor
Gly46 (main-N) Gly45A(E) (main-N) Its backbone amide forms an oxyanion hole to stabilise the transition state. electrostatic stabiliser
Lys34 Lys33A(E) It forms a hydrogen bond with the hydroxyl group oxygen atom of Thr 1 and provides a positive charge to promote deprotonation of this group in the first step of the reaction. electrostatic stabiliser
Ser125 Ser124A(E) It hydrogen bonds with the Thr 1 N and controls the protonation of Thr 1 N. 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. Bochtler M et al. (1997), Proc Natl Acad Sci U S A, 94, 6070-6074. Crystal structure of heat shock locus V (HslV) from Escherichia coli. DOI:10.1073/pnas.94.12.6070. PMID:9177170.
  2. Marques AJ et al. (2009), Chem Rev, 109, 1509-1536. Catalytic mechanism and assembly of the proteasome. DOI:10.1021/cr8004857. PMID:19265443.
  3. Yoo SJ et al. (1997), FEBS Lett, 412, 57-60. Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease. DOI:10.1016/s0014-5793(97)00742-4. PMID:9257689.
  4. Rohrwild M et al. (1996), Proc Natl Acad Sci U S A, 93, 5808-5813. HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. DOI:10.1073/pnas.93.12.5808. PMID:8650174.

Step 1. N-terminal Thr 1 is deprotonated by its own -NH2 group via a water molecule. This activates Thr 1 to nucleophilically attack the carbon of the carbonyl.

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Catalytic Residues Roles

Residue Roles
Gly45A(E) (main-N) electrostatic stabiliser
Lys33A(E) electrostatic stabiliser
Ser124A(E) electrostatic stabiliser
Thr1A(E) proton donor, nucleophile
Thr1A(E) (N-term) proton acceptor

Chemical Components

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

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then deprotonates the N-terminus of Thr 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys33A(E) electrostatic stabiliser
Gly45A(E) (main-N) electrostatic stabiliser
Ser124A(E) electrostatic stabiliser
Thr1A(E) (N-term) proton donor

Chemical Components

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

Step 3. The N-terminus of Thr 1 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the ester bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys33A(E) electrostatic stabiliser
Gly45A(E) (main-N) electrostatic stabiliser
Ser124A(E) electrostatic stabiliser
Thr1A(E) (N-term) proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates another elimination which results in the release of Thr 1 which now accepts a proton from its N-terminus which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys33A(E) electrostatic stabiliser
Gly45A(E) (main-N) electrostatic stabiliser
Ser124A(E) electrostatic stabiliser
Thr1A(E) proton acceptor
Thr1A(E) (N-term) proton donor
Thr1A(E) nucleofuge

Chemical Components

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

Step 1. N-terminal Thr 1 is deprotonated by its own -NH2 group via a water molecule. This activates Thr 1 to nucleophilically attack the carbon of the carbonyl.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then deprotonates the N-terminus of Thr 1.

Step 3. The N-terminus of Thr 1 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the ester bond.

Step 4. The oxyanion initiates another elimination which results in the release of Thr 1 which now accepts a proton from its N-terminus which returns the enzyme to its native state.

Products.

Contributors

Mei Leung, Gemma L. Holliday, Charity Hornby