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Mechanism of lysozyme

Lysozyme digests bacterial cell walls by breakingb(1- 4) glycosidic bonds between (N- acetylmuramicacid (NAM) and N-acetylglucosamine (NAG)

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The interactions of lysozyme with its

substrate

View of the binding cleft with the substrate

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The Phillips mechanism

1. Lysozyme attaches to a bacterial cell wall by binding to ahexasaccharide 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 oxoniumion 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 mechanismwith 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.

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Serine proteases

P-Nitrophenolate is veryyellow while the acetate is

colorless. This is an example

of an artificial substrate!

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The kinetics show

1. A burst phase where the product is rapidlyformed with amounts stoichiometric with theenzyme.

2. Slower steady state that is independent of

substrate concentration.

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A covalent bond between a Serine and the substratesuggests an active Serine. These Serines can be

labeled with inhibitors such as diidopropyl

phosphofluoridate specifically killing the enzyme.

Ser 195 is specifically labeled

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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.

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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)

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The reaction of TPCK with His 57 of chymotrypsin

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Bovine

Trypsin

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Bovine trypsin

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The catalytic triad

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Catalytic mechanism

1. After the substrate binds Ser 195 nucleophilicallyattacks 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 ofcatalysis is only 0.05% of wild-type.

2. Tetrahedral intermediate decomposes to the acyl-

enzyme intermediate. His 57 acts as an acid donating aproton.

3. The enzyme is deacylated by the reverse of step 1 with

water the attacking nucleophile and Ser 195 as the leaving

group.

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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.

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Triad charge transfer complex stabilization