Mechanism Of Lysozyme

Question 1

What is a lysozyme?

Answer 1

It is a part of an organism defence against bacterial invasion

It is part of the innate immune system (the immune system you are born with)

Question 2

Where can you find lysozyme?

Answer 2

This is an enzyme, isolated in tears and bodily fluids and in the whites of eggs

Question 3

Why do you study lysozyme?

Answer 3

It was the first enzyme structure to have its 3-D structure determined
Highlights the need to use many different experiments to elucidate mechanism
Brings together many concept- structure, substrate binding, use of isotopes, general acid and base hydrolysis, mechanistic ideas, TS stabilisation and strain

Question 4

What is glycoside (acetal) hydrolysis?

Answer 4

What is added and OR is the leaving group
An acetal forms a hemiacetal
It is catalysed by glycosyl hydrolyse which chops up sugars

Question 5

What is an important role of glycosyl hydrolase? (Lysozyme)

Answer 5

It chops up sugars
Importantly it breaks down peptidoglycan in the cell wall of bacteria
The breaks down the structural stability
Breaks glycosidic bond

Question 6

What provides a bacteria cell wall with protection?

Answer 6

Polymers of sugars are cross linked by peptides which provides protection for bacteria cell wall

Question 7

What is the use of 18OH2?

Answer 7

Isotopic labelling experiment

Question 8

What is the isotopic labelling experiment?

Answer 8

Treat peptide chain with 18OH2 in the presence of lysozyme
Water acts as a nucleophile as it is in the product
Retention of configuration
There is no inversion
(See mechanism)

Question 9

How can you tell the difference between beta and alpha using NMR?

Answer 9

The couple contain between H1 and H2 has an angular dependence
The trans diaxial angle (180) gives a large j1,2 8Hz- this is beta
The angle (90) gives a small j1,2 2Hz- this is alpha

Question 10

What does the pH profile look like.

Answer 10

Classic bell shaped
Maximum activity at pH 5
Most active at pH5
If you j crease or decrease pH from 5 the rate drops
It goes there must be 2 residues involved in catalysis with pka at 4 and 6
The deprotonated residues are not as good in catalysis

Question 11

How many sugars can lysozyme bind to?

Answer 11

Lysozyme has 6 subsides in its enzyme activate site

These all contribute to catalysis- it wants to bind to 6 sugars which gives the max rate

Question 12

Outline information relating to mechanism of lysozyme

Answer 12

Mechanism involved retention of anomeric configuration
6 sub sites contribute to catalysis
Cleavage between the D and E sugars
PH profile shows dependence of two groups with pkas of 4 and 6

Question 13

Why is the mechanism of acetal hydrolysis not inversion?

Answer 13

SN2 mechanism proved wrong
Product has has net inversion of stereochemistry (back face attack) which is not the product
See mechanism

Question 14

What is the shielded SN1 mechanism of lysozyme ?

Answer 14

Shielded SN1 was proved wrong
Mechanism results in net retention of configuration, not a single SN2 type displacement
See mechanism

Question 15

What is the correct mechanism of lysozyme?

Answer 15

Double displacement
Two subsequential SN2 reactions
Product with net retention of stereochemistry
Use the side chain to accelerate reactions
Covalent intermediate
See mechanism

Question 16

What are the chemical challenges of the lysozyme mechanism?

Answer 16

Unstable glycosyl cation- OR is not a good leaving group

Unlikely alkoxide ion

Question 17

What are the chemical challenges of the lysozyme mechanism overcome?

Answer 17

To stabilise the alkoxide ion, use an acid catalyst to make it neutral and create and better leaving group
Nature uses COOH (general acid catalysis)

Question 18

How does pH affect the acid catalyst for mechanism?

Answer 18

When the acid catalyst is deproonated, the rate is slower

When pH gets above the pka of the residue, it deprotonated the acid

Question 19

What are the two residues involved in the catalysis?

Answer 19

Asp52 sits below
Glu35 sits above (pk6)- this is the catalytic acid as it is protonated
Identified by crystallography
Below pk4, asp52 is protonated

Question 20

What provides the fastest rate (structure of the residues)

Answer 20

For fastest rate
- glu35 is protonated and asp52 is deprotonated which acts as the catalytic nucleophile
Asp52 pka is 4 (becomes deprotonated
Glu35 pka6 (becomes deprotonated)
Therefore fastest rate between 4 and 6 pH

Question 21

How does asp52 act as a nucleophile?

Answer 21

Position below ring to attack- close
Held in correct orientation for attack by enzyme
Proximity and orientation increase rate of attack

Question 22

How does the enzyme change the starting material to increase the rate?

Answer 22

Also enzyme strains the starting material to increase reaction rate- release of strain drives the reaction forwards

Question 23

Outline the overall mechanism of generic double displacement

Answer 23

COO- residues nucleophile (asp) attacks from below and acid COOH (glu) attacks from above, SN2- HOR leaving group
Covalent intermediate with inversion of configuration - SN2 (base attack)
Overall retention of configuration
See mechanism

Question 24

Describe the reaction coordinate of enzymes

Answer 24

Enzyme substrate complex
Enzyme destabilised the starting material 1st SN2
Enzyme intermediate 
2nd SN2
Enzyme product complex is formed

Question 25

Describe the TS of the generic double displacement intermediate

Answer 25

Extra O in the ring
Add partially positive charge to the carbon
1) sp2 hybridisation
2) positive charge
See diagram

Question 26

What should inhibitors mimic?

Answer 26

They should mimic the transition state as enzymes bind to these the best

Question 27

Outline lysozyme catalystis in a nutshell- summary

Answer 27

Acid base catalysis- glu35 as an acid then base
Covalent catalysis- asp52 as nucleophile
Stress and strain- substrate distortion- the enzyme distorts the substrate towards the TS conformation
Inhibition by transition state mimics