8: LYSOZYMES

Question 1

role of lysozymes

Answer 1

• destroy peptidoglycan bacterial cell walls by hydrolysing the ß1-4 glycosidic linkage between NAM and NAG sugar units
• often found as a natural antibiotic e.g. in hen egg whites
• produced in tear ducts to keep eyes clean
• involved in hydrolysing polyNAG in chitin of fungi cell walls

Question 2

structure of NAM/NAG

Answer 2

• sugar units are cyclic
• derivatives of glucose except that in C2 theres NH2 instead of OH and an acetyl derivative (CH3-C=O) in NAG
• NAM has N-acetyl muramic acid in C3

Question 3

lysozyme structure

Answer 3

• small protein (-12 kD)
• Asp52 and Glu35 are key residues that play a role in catalysis; found in either side of glycosidic linkage (at site where hydrolysis occurs)
• glycosidic linkage is broken because polysaccharide binds into this groove to form ES complex

Question 4

discovery of lysozymes

Answer 4

• predicted from model building studies that there was a space for 6 different sugar units to bind into groove (labelled A, B, C, D, E, F)
• phillips crystallised the lysozyme w/small fragments; based on those structures he was able to postulate a model for how polysaccharides could bind into the surface of lysozyme w/respect to asp/glu
• use oligosaccharides as substrates; rate of catalysis high only w/ large oligosaccharides

Question 5

binding at sub-sites of lysozyme

Answer 5

• by looking at free energies of binding of monosaccharides to each site, you could determine whether binding was favorable to particular site or not
• A has -ve ^G of binding so has favorable interaction; all except subsite D (+ve) so sugar is being strained from going into D
• the favorable binding offsets the unfavorable binding to site D
• sugar in D adopts a half-chair conformation (energetically less favorable; O down/C up)
• chair conformation does not fit into D because of steric clashes
• change in conformation involves structural distortion and requires energy driven by the favorable binding of other sugars into other subsites

Question 6

hydrolysis of polysaccharide

Answer 6

• hydrolyse acetal (SN1-type-mechanism)
  1. protonation of O to make better LG
  2. bond breaks and R’’ (next sugar) leaves to form carbocation; favoured because next door O can delocalise +ve charge to form oxonium ion so have resonance stabilisation
  3. have Nu (OH) to bind to carbocation and form hemiacetly product

Question 7

how to have good resonance stabilisation

Answer 7

• make sure p-prbital on O and C are at same level to get good overlap to generate pi-bond in half-chair conformation
• D-ring oxonium ion intermediate in Phillips mechanism is stabilised by resonance in half-chair but not chair

Question 8

2 possible mechanisms explaining activity of lysozyme

Answer 8

• reaction proceeds w/retention of configuration = OH occupies same place as original O linkage between two sugars
• koshland SN2
• Phillips SN1

Question 9

Phillips SN1 Mechanism

Answer 9

1. Glu35 acts as gen. acid and protonates the bridge O of glycosidic bond; usually high pka to ensure protonated state
2. ejection of protonated LG; LP on O attacks C which disrupts octet rule and move LP onto O; O- is not good LG so needs to protonate to form first product OH (H comes from Glu)concerted
3. glycosidic bond cleaved leaving a +ve D-ring oxonium ion which is stabilised by favorable electrostatic interaction w/-ve Asp52 carboxylate and enzyme-induced distortion of the D-ring to enhance resonance stabilisation
4. Glu35 in deprotonated state acts as gen.base to activate water to perform addition at carbocation
5. OH- attacks C, Asp52 is deprotonated
   role of Asp52: plays electrostatic role to stabilise formation of carbocation and oxonium ion to reduce ^G# ; it sterically hinders the active site so OH- can only attack from one direction (explains retention of configuration)

Question 10

Koshland SN2-type mechanism

Answer 10

-to retain configuration have to have 2 back-to-back SN2 steps because 1x goes with inversion of configuration

1. -ve Asp52 carboxylate acts as Nu to displace first product
2. concerted mechanism Glu35 acts as gen. acid and protonates bridge O in glycosidic bond (Glu35 has high pKa to ensure protonated state); reaction is aided by distortion of D-ring
3. glycosyl-enzyme covalent intermediate is formed (covalent catalysis); covalent bond between sugar and Asp52 (inversion of configuration)
4. Glu35 in deprotonated state acts as gen.B to activate water to perform Nu attack SN2 reaction to displace Asp52 to release second product