Lysozyme (glycosyl hydrolase 22 family)

 

Lysozymes have primarily a bacteriolytic function, it hydrolyses specific bonds between NAG and NAM in peptidoglycan, which forms the bacterial cell wall. Lysosymes in tissues and body fluids are associated with the monocyte- macrophage system and enhance the activity of immunoagents.

Historically there has been much debate about the mechanism of the enzyme, but recent evidence overwhelmingly supports the presence of a covalently bound intermediate. This discredits the Phillips mechanism which proposed an oxycarbenium intermediate.

 

Reference Protein and Structure

Sequence
P00698 UniProt (3.2.1.17) IPR000974 (Sequence Homologues) (PDB Homologues)
Biological species
Gallus gallus (Chicken) Uniprot
PDB
1dpx - STRUCTURE OF HEN EGG-WHITE LYSOZYME (1.65 Å) PDBe PDBsum 1dpx
Catalytic CATH Domains
1.10.530.10 CATHdb (see all for 1dpx)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.17)

water
CHEBI:15377ChEBI
+
beta-MurNAc-(1->4)-beta-D-GlcpNAc
CHEBI:87004ChEBI
→
N-acetyl-beta-D-muramic acid
CHEBI:40729ChEBI
+
N-acetyl-beta-D-glucosamine
CHEBI:28009ChEBI
Alternative enzyme names: 1,4-N-acetylmuramidase, N,O-diacetylmuramidase, L-7001, PR1-lysozyme, Globulin G, Globulin G1, Lysozyme g, Mucopeptide N-acetylmuramoylhydrolase, Mucopeptide glucohydrolase, Muramidase,

Enzyme Mechanism

Introduction

Asp52 attacks the C1 of the peptidoglycan in a nucleophilic substitution that results in the NAG portion of the peptidoglycan being covalently attached to the enzyme and the NAM being released. NAM deprotonates Glu35. Glu35 deprotonates water, which attacks the C1 of the covalently bound NAG intermediate in a nucleophilic substitution that results in the NAG product and free Asp52

Catalytic Residues Roles

UniProt PDB* (1dpx)
Asp66, Ser68, Asn64, Asn77 Asp48A, Ser50A, Asn46A, Asn59A Part of a hydrogen bonding network which is important for catalytic activity, not shown in the mechanism as these residues are not directly involved in catalysis.
Asp70 Asp52A Asp52 is the nucleophile for the first stage of the reaction, attacking C1 of the peptidoglycan. It is covalently bound to the substrate in the intermediate. covalently attached, nucleofuge, nucleophile, polar/non-polar interaction
Glu53 Glu35A Having been deprotonated by the substrate, Glu35 deprotonates water to activate it as a nucleophile. hydrogen bond acceptor, hydrogen bond donor, 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

bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, intermediate formation, overall product formed, overall reactant used, rate-determining step, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate collapse, intermediate terminated, hydrolysis

References

  1. Vocadlo DJ et al. (2001), Nature, 412, 835-838. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. DOI:10.1038/35090602. PMID:11518970.
  2. Kawaguchi Y et al. (2015), Biosci Biotechnol Biochem, 79, 196-204. Asp48 function in the hydrogen-bonding network involving Asp52 of hen egg-white lysozyme. DOI:10.1080/09168451.2014.963502. PMID:25514638.
  3. Held J et al. (2014), Acta Crystallogr D Biol Crystallogr, 70, 1136-1146. The active site of hen egg-white lysozyme: flexibility and chemical bonding. DOI:10.1107/S1399004714001928. PMID:24699657.
  4. Bottoni A et al. (2005), Proteins, 59, 118-130. A theoretical DFT investigation of the lysozyme mechanism: Computational evidence for a covalent intermediate pathway. DOI:10.1002/prot.20396. PMID:15688446.
  5. Kirby AJ (2001), Nat Struct Biol, 8, 737-739. The lysozyme mechanism sorted -- after 50 years. DOI:10.1038/nsb0901-737. PMID:11524668.

Step 1. During this step, the C1 migrates (in an electrophilic manner) from above the ring plane to below the ring plane to approach the catalytic nucleophile (Asp52), which hardly changes its position during the course of the reaction. Asp52 then attacks the C1 of the peptidoglycan in a nucleophilic substitution that results in the NAG portion of the peptidoglycan being covalently attached to the enzyme and the NAM being released. NAM deprotonates Glu35.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu35A hydrogen bond donor
Asp52A polar/non-polar interaction
Glu35A proton donor
Asp52A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, intermediate formation, overall product formed, overall reactant used, rate-determining step

Step 2. During this step, the C1 migrates from its position below the ring plane to form a bond with a water molecule positioned in the location previously occupied by the substrate glycosidic oxygen [PMID:11518970]. Glu35 deprotonates water, which attacks the C1 of the covalently bound NAG intermediate in a nucleophilic substitution that results in the NAG product and free Asp52.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu35A hydrogen bond acceptor
Asp52A covalently attached
Glu35A proton acceptor
Asp52A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate collapse, intermediate terminated, overall product formed, hydrolysis
Download: Image, Marvin File

Step 1. During this step, the C1 migrates (in an electrophilic manner) from above the ring plane to below the ring plane to approach the catalytic nucleophile (Asp52), which hardly changes its position during the course of the reaction. Asp52 then attacks the C1 of the peptidoglycan in a nucleophilic substitution that results in the NAG portion of the peptidoglycan being covalently attached to the enzyme and the NAM being released. NAM deprotonates Glu35.

Step 2. During this step, the C1 migrates from its position below the ring plane to form a bond with a water molecule positioned in the location previously occupied by the substrate glycosidic oxygen [PMID:11518970]. Glu35 deprotonates water, which attacks the C1 of the covalently bound NAG intermediate in a nucleophilic substitution that results in the NAG product and free Asp52.

Products.

Introduction

The so-called Phillips mechanism in which the enzyme proceeds via a oxycarbenium intermediate. Text book mechanism of Lysozyme, disproved.

Catalytic Residues Roles

UniProt PDB* (1dpx)
Asp70 Asp52A Activate and stabilise the carbocation intermediate formed. electrostatic stabiliser
Glu53 Glu35A Acts as a general acid/base. 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

unimolecular elimination by the conjugate base, proton transfer, bimolecular nucleophilic addition

References

  1. Davies G et al. (1995), Structure, 3, 853-859. Structures and mechanisms of glycosyl hydrolases. DOI:10.1016/s0969-2126(01)00220-9. PMID:8535779.

Step 1. The anomeric oxygen eliminates the NAG with concomitant deprotontaion of Glu35.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp52A electrostatic stabiliser
Glu35A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer

Step 2. Glu35 deprotonates the catalytic water, which adds to the carbon of the oxycarbocation group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp52A electrostatic stabiliser
Glu35A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition
Download: Image, Marvin File

Step 1. The anomeric oxygen eliminates the NAG with concomitant deprotontaion of Glu35.

Step 2. Glu35 deprotonates the catalytic water, which adds to the carbon of the oxycarbocation group.

Products.

Contributors

Gemma L. Holliday, Sophie T. Williams, Emma LR Compton, Craig Porter, Anna Waters, Carine Berezin, James Willey