Lec 6 - Lysozyme & Glycosidases

Question 1

What is the main role of lysozymes?

Answer 1

• first line of defence against bacterial attack because they cleave peptidoglycan

Question 2

Which type of bacteria (Gram +/-ve) is lysozyme effective against and draw its structre

Answer 2

Gram +ve

Question 3

Where does lysozyme cleave the peptidoglycan chain and what points of the chain does it avoid and why?

Answer 3

after NAM molecules and before NAGs

avoids parts of the chains that have crosslinks - avoids steric clashes

Question 4

Give the NAM-NAG linkage full name (like which Cs are included in the bond) and draw a rough outline of its structure - including where lysozyme attacks

Answer 4

NAM B(1-4) NAG linkage

Question 5

At which sites in the active sites of the lysozyme does cleavage occur and name the residues involved in this cleavage

Answer 5

sites D (NAM) and E (NAG)

Asp52 and Glu35

Question 6

Draw the full structure of the lysozyme enzyme

Answer 6

Question 7

Why is Glu35 in its protonated form (when its natural pKa is around 4 and lysozymes optimal pH is 6 suggesting that it should be deprotonated)?

Answer 7

Glu35 is surrounded by hydrophobic residues therefore in uncharged, protonated form

Question 8

How was the correct acyl-enzyme lysozyme mechanism discovered?

Answer 8

used a F substrate analogue that formed a stable compound that could be isolated. also mutated Glu -> Gln therefore could not take part in the reaction

Question 9

Use this rough outline of the reaction mechanism to fill in the arrows

Answer 9

Question 10

Give the name of the 4 other glycosidases studied and describe the reactions they catalyse

Answer 10

• cellulases - breakdown of cellulose into glucose
• amylases - breakdown starch into the various monomers that make up starch (eg can have 2 maltose)
• neuraminidases - break up haemagglutinin bonded to sialic acid on glycoproteins of host cells
• lactases - break up lactose -> glucose & galactose

Question 11

Whilst the structures of these glycosidases may vary what do they all have in common

Answer 11

both use carboxylate side chains (Asp and Glu) acting as an acid/base

Question 12

Complete the mechanism catalysed by a amylase

Answer 12

Question 13

Describe the structure of the lactase enzyme

Answer 13

a very large enzyme

homotetramer

mutations w/in this enzyme can result in a lactose intolerance

Question 14

Draw a rough structure of the flu virus

Answer 14

Question 15

Describe how the virus attaches to the host cell and how the action of neuraminidase removes this interaction

Answer 15

• virus attaches via haemagluttin binding to sialic acid groups on glycoproteins of the host cell
• NMase catalyses the breaking of the glycosidic bond that has formed and therefore allows the virus to detach from the infected host cell

Question 16

Describe the action of Tamiflu as an antiviral. State another class of inhibitors that stop the release of viral particles from host cell membranes

Answer 16

it is a neuraminidase inhibitor resulting in viral aggregation @ host cell surface

sialic acid mimic drugs are also in use to bind to NMase and therefore block its AS