LYSOZYMELYSOZYMEMECHANISM OF MECHANISM OF

ACTIONACTION

D.YESHWANTH KUMAR(1st YEAR M.TECH BIOTECHNOLOGY)

INTRODUCTIONINTRODUCTIONNatural Anti-bacterial agent

129 amino acid residues

Molecular Mass – 14.3 kD (hen egg lysozyme)

Found at: egg whites and tears

Substrate: Peptidoglycan

3-D Structure: Determined by David Phillips, et al. in 1965

SUBSTRATE: PEPTIDOGLYCANSUBSTRATE: PEPTIDOGLYCAN

Site of cleavage

• It’s a Carbohydrate• NAM: N-acetylmuramic Acid• NAG: N-acetylglucosomine• Cleaves NAM-NAG site at β(1→4)

glycosidic C–O bond• NAM contains lactyl (lactic acid)

group side Chain which forms the peptide bridge

STRUCTURE OF LYSOZYME (RIBBON DIAGRAM)

•The Blue sticks are Glu35 and Asp52 (active site)

•The red sticks are the substrate (peptidoglycan)

LYSOZYME + SUBSTRATE LYSOZYME + SUBSTRATE REACTIONREACTION

KEY

• RO: Lactyl group

• NAc/AcN: N-acetyl group

LYSOZYME: MECHANISM OF LYSOZYME: MECHANISM OF ACTION (SACTION (SNN1 MECHANISM)1 MECHANISM)

Peptidoglycan binds to the active site of the lysozyme

This reaction is a nucleophilic displacement reaction

Half life of the intermediate 10-12 secnds

Acid Catalysis

Highly Unstable

LYSOZYME: MECHANISM OF LYSOZYME: MECHANISM OF ACTION (SACTION (SNN1 MECHANISM)1 MECHANISM)

Proposed in 1965

This mechanism accepted for around 3 decades

Reasons for Failure:

Experimentally not proved and satisfactory

Lysozyme catalyzes the reaction where highly unstable intermediates are being formed

LYSOZYME: MECHANISM OF LYSOZYME: MECHANISM OF ACTION (SACTION (SNN2 MECHANISM)2 MECHANISM)

Involves two consecutive direct displacement steps

Acid catalysis addressed as in first step

More stable covalent intermediate was formed

This also involves nucleophilic displacement reaction

Acid Catalysis

Covalent Catalysis

LYSOZYME: MECHANISM OF LYSOZYME: MECHANISM OF ACTION (SACTION (SNN2 MECHANISM)2 MECHANISM)

Proposed in 2001

Proven by Stephen Withers, et al.

Experimentally proven

Formation of more stable covalent intermediate (covalent catalysis)