Lysozyme

Question 1

name the 2 major forms of lysozymes

Answer 1

Human lysozyme in tears
Hens Egg White (HEW) Lysozyme in Eggs

Question 2

what is the substrate of this enzyme

Answer 2

peptidoglycan

Question 3

what is the non-competitive inhibitor of this enzyme

Answer 3

lipopolysaccharide - has highly favoured binding to lysozyme

Question 4

how does this enzyme act upon peptidoglycan

Answer 4

catalytically cleaves peptidoglycan @ active site -> smaller units [E+S-> E+P]

Question 5

what part of the body does the enzyme effect

Answer 5

Hypothalamus

Question 6

it has a similar mode of action to many other ? enzymes

Answer 6

depolymerizing enzymes [breaking down polymerase]

Question 7

what specifically does the enzyme act upon in the peptidoglycan layer of bacteria

Answer 7

catalyzes the hydrolysis of the b(1-4) glycosidic links between N-acetylmuramic acid (NAM) and the N-acetylglucosamine (NAG) residues of the peptidoglycan layer in bacteria.

Question 8

bacterial cell wall is composed of peptidoglycan which is made of alternating

Answer 8

N- acetylglucosamine (NAG or GlcNAc) and
N- acetylmuramic acid (NAM or MurNAc)

Question 9

the sugar units in peptidoglycan is joined by? and cross-linked by ? which does what

Answer 9

β-1-4 glycosidic bond
peptide bridges which stabilises molecules

Question 10

peptide bridges contain?

Answer 10

non-standard L-form and D-form of A.A

Question 11

how many A.A. does it have

Answer 11

129

Question 12

what word is used -> describe the shape

Answer 12

ellipsoid shape [i.e. squashed circle]

Question 13

what are the 2 domains

Answer 13

alpha helical, 3 stranded anti-parallel sheet + 2 helices

Question 14

what type of bonds are present

Answer 14

4 disulphide bonds - 2 in each domain

Question 15

is NAG3 hydrolyzable

Answer 15

non-hydrolyzable- competitive inhibitor i.e. binds -> same site as substrate

Question 16

the cleft in the enzymes active site van be completely occupied by how many sugar residues

Answer 16

6

Question 17

what determines the substrate binding

Answer 17

a number of H bonding interactions

Question 18

what was the most abundant product from the catalyzed hydrolysis of a 6 unit substrate using this enzyme- this confirms the cleavage occurs where

Answer 18

(ABCD)4 and (EF)2
NAM [D residue] and NAG [E residue]
NAG(A)-NAM(B)-NAG -NAM(D)-NAG(E) –NAM(F)

Question 19

THE PHILIPS MECHANISM
Modelling further showed that two x residues are near hydrolyzed bond (Asp52 and Glu35). [@ Position y and z in the 129 amino acid chain]
At optimum pH for lysozyme (pH q ), Asp52 (pKa x) would be de-protonated, and Glu35 (pKa y) protonated.
NB pKa corresponds to the pH at which the concentration of x and y forms are equal

Answer 19

x = acidic
y = 52 and z= 35
q = 5
pka 3.5
pka 6.3
ionised and neutral forms

Question 20

Mechanism of Lysozyme acc-> Philips model
Mechanism (Phillips Model)
1) The x group of Glu 35 donates a H+ to the glycosidic oxygen between the D and E ring.
2) This creates a Y charge on the C1 of the D ring (a carbonium ion).
3) The E and F residues are broken off and hence x away.
4) The carbonium ion intermediate then reacts with y- from the solvent (H2O) and Glu 35 becomes x
The ABC&D residues diffuse leaving the enzyme ready to work again.

Key features of the enzymatic mechanism
a) Acid base catalysis
b) Carbonium ion formation stabilised by Asp 52 (negative charged carboxylate stabilises carbonium group)
c) Distortion and strain on the bound peptidoglycan. Focused between D & E residues

Answer 20

x = -COOH
Y = positive
x = diffuse
y = -OH
x = protonated