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Mechanism of Lysozyme

Lysozyme digests bacterial cell walls by breaking bonds between N-acetylmuramic acid and N-acetylglucosamine. It binds to a hexasaccharide unit and uses an acid catalyst and stabilization of an oxonium ion to cleave bonds via SN2 mechanism. Serine proteases use a catalytic triad of Serine, Histidine, and Aspartic acid to form a tetrahedral intermediate that is then hydrolyzed to release the substrate.

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Raju Gangadharan
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36 views

Mechanism of Lysozyme

Lysozyme digests bacterial cell walls by breaking bonds between N-acetylmuramic acid and N-acetylglucosamine. It binds to a hexasaccharide unit and uses an acid catalyst and stabilization of an oxonium ion to cleave bonds via SN2 mechanism. Serine proteases use a catalytic triad of Serine, Histidine, and Aspartic acid to form a tetrahedral intermediate that is then hydrolyzed to release the substrate.

Uploaded by

Raju Gangadharan
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Mechanism of lysozyme

Lysozyme digests bacterial cell walls by breaking b(1- 4) glycosidic bonds between (N- acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)

The interactions of lysozyme with its substrate


View of the binding cleft with the substrate

The Phillips mechanism


1. Lysozyme attaches to a bacterial cell wall by binding to a hexasaccharide unit. The D residue is distorted towards the half-chair. 2. Glu 35 transfers its proton to the O1 of the D ring (general acid catalysis) C1-O1 bond is cleaved generating an oxonium ion at C1. 3. Asp 52 stabilizes the oxonium ion through charge-charge interactions. The carboxylate can not form a covalent bond because distances are too great. Reaction via a SN2 mechanism with transient formation of a C --O bond to the enzyme. 4. E ring group is released from the enzyme yielding a glycosyl-enzyme intermediate which adds water to reverse the chemistry and reprotonate Glu 35.

Serine proteases

P-Nitrophenolate is very yellow while the acetate is colorless. This is an example of an artificial substrate!

The kinetics show 1. A burst phase where the product is rapidly formed with amounts stoichiometric with the enzyme. 2. Slower steady state that is independent of substrate concentration.

A covalent bond between a Serine and the substrate suggests an active Serine. These Serines can be labeled with inhibitors such as diidopropyl phosphofluoridate specifically killing the enzyme. Ser 195 is specifically labeled

DIPF is extremely toxic because other active Serines can be labeled. Such as acetylcholine esterase.

Nerve gases, serin gas, are very toxic!! Many insecticides also work this way.

Affinity labeling
His 57 is a second important catalytic residue. A substrate containing a reactive group binds at the active site of the enzyme and reacts with a nearby reactive amino acid group. A Trojan horse effect.

Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

The reaction of TPCK with His 57 of chymotrypsin

Bovine Trypsin

Bovine trypsin

The catalytic triad

Catalytic mechanism
1. After the substrate binds Ser 195 nucleophilically attacks the scissile peptide bond to form a transition state complex called the tetrahedral intermediate (covalent catalysis) the imidazole His 52 takes up the proton Asp 102 is hydrogen bonded to His 57. Without Asp 102 the rate of catalysis is only 0.05% of wild-type. 2. Tetrahedral intermediate decomposes to the acylenzyme intermediate. His 57 acts as an acid donating a proton. 3. The enzyme is deacylated by the reverse of step 1 with water the attacking nucleophile and Ser 195 as the leaving group.

1. Conformational distortion forms the tetrahedral intermediate and causes the carboxyl to move close to the oxyanion hole 2. Now it forms two hydrogen bonds with the enzyme that cannot form when the carbonyl is in its normal conformation. 3. Distortion caused by the enzyme binding allows the hydrogen bonds to be maximal.

Triad charge transfer complex stabilization

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