Assemblin

 

Herpesviruses, such as human cytomegalovirus, are large double stranded DNA viruses that infect most species throughout the animal kingdom. They encode a protease that is essential for production of infectious virons: this enzyme catalyses the maturational processing of the herpesvirus assembly protein.

Human cytomegalovirus (HCMV) protease is a serine protease (member of the peptidase S21 family) that is involved in the proteolytic processing of the assembly protein precursor during capsid maturation. CMV protease shares no sequence homology with classical serine proteases and contains a Ser-His-His catalytic triad, rather than the classical Ser-His-Asp/Glu. The mechanism, however, is much the same. HCMV protease is involved in autoproteolytic cleavage at two sites to produce an enzyme with full catalytic activity. The enzyme cleaves between Ala-Ala/Ser. HCMV protease exists in a monomer-dimer equilibrium, with the homodimer being active and the monomer not. HCMV protease is a potential antiviral drug target.

 

Reference Protein and Structure

Sequence
P16753 UniProt (3.4.21.97) IPR001847 (Sequence Homologues) (PDB Homologues)
Biological species
Human herpesvirus 5 strain AD169 (Human cytomegalovirus) Uniprot
PDB
1wpo - HYDROLYTIC ENZYME HUMAN CYTOMEGALOVIRUS PROTEASE (2.0 Å) PDBe PDBsum 1wpo
Catalytic CATH Domains
3.20.16.10 CATHdb (see all for 1wpo)
Click To Show Structure

Enzyme Reaction (EC:3.4.21.97)

water
CHEBI:15377ChEBI
+
Ala-Ser
CHEBI:73394ChEBI
→
L-serine
CHEBI:17115ChEBI
+
L-alanine
CHEBI:16977ChEBI

Enzyme Mechanism

Introduction

Herpesvirus protease uses a Ser-His-His catalytic triad. Ser 132 acts as a nucleophile to attack the peptide bond, while His 63 deprotonates the attacking serine residue. The resulting tetrahedral intermediate is stabilised by an oxyanion hole consisting of the backbone NH of Arg 165 and two water molecules positioned by the guanidinium group of Arg 166. Collapse of the tetrahedral intermediate with protonation of the departing amine by His 63 generates an acyl-enzyme intermediate. This is then hydrolysed by a water molecule that is deprotonated by His 63. His 157 functions to modify the pKa of His 63, although its effect of catalysis is much smaller than that of the aspartate in the classical Ser-His-Asp serine proteases.

Catalytic Residues Roles

UniProt PDB* (1wpo)
His63 His63A His63 deprotonates Ser132 to enhance its nucleophilicity and protonates the amine leaving group. It then deprotonates water for attack on the acyl-enzyme intermediate and protonates Ser132 when it acts as a leaving group. His63 forms a hydrogen bond to His157 and the latter stabilises His63 during the catalytic cycle. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His157 His157A His157 forms a hydrogen bond to His63, increasing its pKa, and stabilises the residue during the catalytic cycle. His157 is exposed to the solvent and this limits its functional importance relative to the buried acidic residue in classical serine proteases. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
Ser132 Ser132A Ser132 is deprotonated by His63 and then acts as the nucleophile for attack on the carbonyl carbon of the substrate. Upon intermediate collapse Ser132 forms part of an acyl-enzyme intermediate. During the hydrolysis of this intermediate Ser132 acts as the leaving group and is protonated by His63. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, nucleophile, proton donor, proton acceptor
Ser134 Ser134A Ser134 is thought to stabilise His157 during the catalytic cycle. increase basicity, hydrogen bond donor, increase acidity
Arg166 Arg166A Arg166 is part of the oxyanion hole and forms an indirect hydrogen bond to the oxyanion through water. hydrogen bond donor, electrostatic stabiliser
Arg165 (main-N) Arg165A (main-N) Backbone NH forms part of oxyanion hole that stabilises the tetrahedral intermediate. hydrogen bond donor, 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, hydrolysis, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated

References

  1. Chen P et al. (1996), Cell, 86, 835-843. Structure of the Human Cytomegalovirus Protease Catalytic Domain Reveals a Novel Serine Protease Fold and Catalytic Triad. DOI:10.1016/s0092-8674(00)80157-9. PMID:8797829.
  2. Polgár L (2005), Cell Mol Life Sci, 62, 2161-2172. The catalytic triad of serine peptidases. DOI:10.1007/s00018-005-5160-x. PMID:16003488.
  3. Khayat R et al. (2003), Biochemistry, 42, 885-891. Structural and Biochemical Studies of Inhibitor Binding to Human Cytomegalovirus Protease†. DOI:10.1021/bi027045s. PMID:12549906.
  4. Khayat R et al. (2001), Biochemistry, 40, 6344-6351. Investigating the Role of Histidine 157 in the Catalytic Activity of Human Cytomegalovirus Protease†. DOI:10.1021/bi010158b. PMID:11371196.
  5. Liang PH et al. (1998), Biochemistry, 37, 5923-5929. Site-Directed Mutagenesis Probing the Catalytic Role of Arginines 165 and 166 of Human Cytomegalovirus Protease. DOI:10.1021/bi9726077. PMID:9558326.
  6. Shieh HS et al. (1996), Nature, 383, 279-282. Three-dimensional structure of human cytomegalovirus protease. DOI:10.1038/383279a0. PMID:8805708.
  7. Qiu X et al. (1996), Nature, 383, 275-279. Unique fold and active site in cytomegalovirus protease. DOI:10.1038/383275a0. PMID:8805707.
  8. Cox GA et al. (1995), J Virol, 69, 4524-4528. Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad. PMID:7769716.
  9. Holwerda BC et al. (1994), J Biol Chem, 269, 25911-25915. Activity of two-chain recombinant human cytomegalovirus protease. PMID:7929296.
  10. Welch AR et al. (1993), J Virol, 67, 7360-7372. Herpesvirus proteinase: site-directed mutagenesis used to study maturational, release, and inactivation cleavage sites of precursor and to identify a possible catalytic site serine and histidine. PMID:8230459.

Step 1. His63 deprotonates Ser132, which can then act as the nucleophile for attack on the carbonyl carbon of the substrate. This forms a tetrahedral intermediate that is stabilised by the oxyanion hole. Arg165 and Arg166 form the oxyanion hole. Arg165 forms a direct hydrogen bond to the oxyanion while Arg166 forms an indirect hydrogen bond involving water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg165A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser134A increase basicity, hydrogen bond donor
Arg166A hydrogen bond donor, electrostatic stabiliser
His63A hydrogen bond acceptor, hydrogen bond donor
Ser132A hydrogen bond donor
His157A hydrogen bond acceptor, electrostatic stabiliser, increase basicity
His63A proton acceptor
Ser132A proton donor, nucleophile

Chemical Components

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

Step 2. The tetrahedral intermediate collapses and eliminates the new N-terminus of the protein, which is then protonated by His63.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg165A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser134A increase acidity, hydrogen bond donor
Arg166A hydrogen bond donor, electrostatic stabiliser
His63A hydrogen bond donor
Ser132A covalently attached
His157A increase acidity, hydrogen bond acceptor
His63A proton donor

Chemical Components

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

Step 3. His63 deprotonates water, which can then act as the nucleophile for attack on the carbonyl carbon of the acyl-enzyme. This forms a tetrahedral intermediate that is stabilised by the oxyanion hole.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg165A (main-N) electrostatic stabiliser, hydrogen bond donor
Ser134A increase basicity, hydrogen bond donor
Arg166A electrostatic stabiliser, hydrogen bond donor
His63A hydrogen bond acceptor, hydrogen bond donor
Ser132A covalently attached
His157A increase basicity, hydrogen bond acceptor, electrostatic stabiliser
His63A proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, hydrolysis, intermediate formation, overall reactant used

Step 4. The tetrahedral intermediate collapses and eliminates Ser132, which is then protonated by His63.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg165A (main-N) hydrogen bond donor, electrostatic stabiliser
Ser134A increase acidity, hydrogen bond donor
Arg166A hydrogen bond donor, electrostatic stabiliser
His63A hydrogen bond donor
Ser132A covalently attached, hydrogen bond acceptor, hydrogen bond donor
His157A increase acidity, hydrogen bond acceptor
His63A proton donor
Ser132A nucleofuge, proton acceptor

Chemical Components

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

Step 1. His63 deprotonates Ser132, which can then act as the nucleophile for attack on the carbonyl carbon of the substrate. This forms a tetrahedral intermediate that is stabilised by the oxyanion hole. Arg165 and Arg166 form the oxyanion hole. Arg165 forms a direct hydrogen bond to the oxyanion while Arg166 forms an indirect hydrogen bond involving water.

Step 2. The tetrahedral intermediate collapses and eliminates the new N-terminus of the protein, which is then protonated by His63.

Step 3. His63 deprotonates water, which can then act as the nucleophile for attack on the carbonyl carbon of the acyl-enzyme. This forms a tetrahedral intermediate that is stabilised by the oxyanion hole.

Step 4. The tetrahedral intermediate collapses and eliminates Ser132, which is then protonated by His63.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, Steven Smith, Charity Hornby