Catalytic Mechanisms and

Catalytic Action of Lysozyme

SCPAA3

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Week 3: Lesson 5
Introduction

In this week you will learn about catalytic mechanisms: The types of catalytic mechanisms
that enzymes employ have been classified as:
1.Acid–base catalysis
2.Covalent catalysis
3.Metal ion catalysis
4.Proximity and orientation effects
5.Preferential binding of the transition state complex
 What will be covered
in today’s lesson?
Contrasting the chemical basis for
acid-base catalysis, covalent catalysis
and metal ion catalysis.
Week 3
Lesson 5 Discussing how enzymes accelerate
reactions through proximity and
orientation effects and by preferential
binding of the transition state

Identifying and motivating through

interpreting diagrams the type of
catalysis shown
Case Study
• [Enter Case Study]
• [Case studies can be used to encourage student engagement in
class]

• Source:

• [slides can be re-ordered to suit purpose]

The types of catalytic mechanisms that enzymes employ have
been classified as:

1. Acid–base catalysis
2. Covalent catalysis
3. Metal ion catalysis
4. Proximity and orientation effects
5. Preferential binding of the transition state complex

5
 Drawing reaction mechanism (Box 11-1)

• We use curved arrow convention to show how pairs of electrons are rearranged
during a reaction.
• Focus on the more common two-electron reactions
• The movement of an electron pair is symbolized by a curved arrow starting at the
electron rich centre pointing towards the electron deficient centre
Bond Breakage:

Bond Forming:

6
 Drawing reaction mechanism (Box 11-1)

Eg. Schiff base reaction:

Tautomers are two molecules with the same molecular formula but
different connectivity - constitutional isomers, in other words - which can
interconvert in a rapid equilibrium. Can be seen in relocation of a proton

© 2017 John Wiley & Sons, Inc. All rights reserved 7

 Acid-base catalysis

Acid–Base Catalysis Occurs by Proton Transfer – Mechanism of Keto-enol tautomerization

Uncatalyzed

Proton transfer from an acid

Acid catalyzed
lowers the free energy of a
reaction’s transition state.

Base catalyzed The rate is increased by proton removal by

a base

8
Figure 11-8 © 2017 John Wiley & Sons, Inc. All rights reserved
Activity

Read and discuss:

Effects of pH on enzyme
activity (Box 11-2)
RNase A Is an Acid–Base Catalyst.
The RNase A mechanism

Figure 11-10 © 2017 John Wiley & Sons, Inc. All rights reserved 11
The RNase A mechanism

Figure 11-10 © 2017 John Wiley & Sons, Inc. All rights reserved 12
The RNase A mechanism

13
Figure 11-10 © 2017 John Wiley & Sons, Inc. All rights reserved
 Covalent catalysis usually requires a nucleophile

Covalent catalysis accelerates reaction rates through the transient formation of a catalyst–
substrate covalent bond.

Covalent catalysis can be conceptually decomposed into three stages:

1. The nucleophilic reaction between the catalyst and the substrate to form a
covalent bond.
2. The withdrawal of electrons from the reaction centre by the now electrophilic
catalyst.
3. The elimination of the catalyst, a reaction that is essentially the reverse of stage
1.

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Biologically important nucleophilic & electrophilic groups

Figure 11-12 © 2017 John Wiley & Sons, Inc. All rights reserved 15
 Biologically important nucleophilic groups

Serine, Tyrosine & Aspartic

Threonine acid &
Glutamic
acid
Cysteine

Lysine

Histidine

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Figure 11-12 © 2017 John Wiley & Sons, Inc. All rights reserved
Biologically important electrophilic groups

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Figure 11-12 © 2017 John Wiley & Sons, Inc. All rights reserved
Decarboxylation of aceto-acetate

Figure 11-11 © 2017 John Wiley & Sons, Inc. All rights reserved 18
Schiff base formation

© 2017 John Wiley & Sons, Inc. All rights reserved 19

 Decarboxylation of aceto-acetate

Uncatalyzed

Elimination of
Nucleophile reaction catalyst

Catalyzed by
primary
amine

Figure 11-11 © 2017 John Wiley & Sons, Inc. All rights reserved 20
 Metal ion cofactors act as catalysts

Metal ions participate in the catalytic process in three major ways:

1. By binding to substrates to orient them properly for reaction.
2. By mediating oxidation–reduction reactions through reversible changes
in the metal ion’s oxidation state.
3. By electrostatically stabilizing or shielding negative charges.

21
Role of Zn2+ in carbonic anhydrase

Figure 11-13 b) © 2017 John Wiley & Sons, Inc. All rights reserved 22
Activity
Discuss how enzymes accelerate reactions through
proximity and orientation effects and by preferential
binding of the transition state
 Catalysis via proximity & orientation

Reaction 1

Reaction 2

© 2017 John Wiley & Sons, Inc. All rights reserved 24

 Geometry of SN2 reaction

By simply binding their substrates, enzymes facilitate their catalyzed reactions in four
ways:

1. Enzymes bring substrates into contact with their catalytic groups. Proximity effects alone can
enhance reaction rates by no more than a factor of 5.
2. Enzymes bind their substrates in the proper orientations for reaction. It is estimated that properly
orienting substrates can increase reaction rates by a factor of up to 100.
3. Charged groups may help stabilize the transition state of the reaction, a phenomenon termed
electrostatic catalysis. The expulsion of water from the active site may enhance this effect. The
charge distribution around the active sites of enzymes may also guide polar substrates toward
their binding site.
4. Enzymes freeze out the relative translational and rotational motions of their substrates and
catalytic groups. This effect can promote rate enhancements of up to 107

25
Geometry of SN2 reaction – binding with proper orientation

Figure 11-14 © 2017 John Wiley & Sons, Inc. All rights reserved 26
Catalysis via preferential transition state binding

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© 2017 John Wiley & Sons, Inc. All rights reserved
 Catalysis via preferential transition state binding

• Enzymes bind the transition state with higher affinity than the substrate
or product

• Enzyme mechanically strain substrates towards transition states (rack

mechanism)

• A good substrate does not need to bind tightly to the enzyme but must
bind tightly when activated to the transition state

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© 2017 John Wiley & Sons, Inc. All rights reserved
Catalysis via preferential transition state binding

29
© 2017 John Wiley & Sons, Inc. All rights reserved
Catalysis via preferential transition state binding

Animation link
Figure 11-15 © 2017 John Wiley & Sons, Inc. All rights reserved 30
Inhibition by transition state analogs

© 2017 John Wiley & Sons, Inc. All rights reserved 31

Week 3: Lesson 6
Introduction

• In this lesson, you will learn about catalytic action of lysozyme.

• Lysozyme is an enzyme responsible for breaking down the cell walls of bacteria.
• It achieves this by catalyzing the hydrolysis of the (1-4) glycosidic linkages between N-acetylmuramic
acid (NAM or MurNAc) and N-acetylglucosamine (NAG or GlcNAc) in the peptidoglycans present in
the bacterial cell wall.
• Additionally, lysozyme can also hydrolyze (1-4)-linked poly(NAG) (known as chitin) , which is a
component of cell walls in most fungi and serves as a major constituent in the exoskeletons of
insects and crustaceans.
 What will be covered
in today’s lesson?
Analyse and discuss the
Week 3 general features of
Lesson 6 substrate binding to
lysozyme.

Discuss and interpret

the catalytic
mechanism of
lysozyme.
Lysozyme cleavage site

Figure 11-16 © 2017 John Wiley & Sons, Inc. All rights reserved
34
Chair & Half-chair conformations

1
2
6

3 5
4

Figure 11-18 © 2017 John Wiley & Sons, Inc. All rights reserved 35
Identification of lysozyme cleavage site

© 2017 John Wiley & Sons, Inc. All rights reserved 36

Non-enzymatic Acid-catalyzed hydrolysis

37
© 2017 John Wiley & Sons, Inc. All rights reserved
Lysozyme reaction mechanism

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Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved
Lysozyme reaction mechanism

1
1

Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved 39
Lysozyme reaction mechanism

1
1

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Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved
Lysozyme reaction mechanism

41
Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved
Lysozyme reaction mechanism

42
Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved
Lysozyme reaction mechanism

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Figure 11-21 © 2017 John Wiley & Sons, Inc. All rights reserved
Outcome 2 – Lysozyme: Transition state analog inhibitor

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Figure 11-22 © 2017 John Wiley & Sons, Inc. All rights reserved
Outcome 2 – Position of D-Ring lysozyme catalysis

Covalent glycosyl-enzyme
intermediate of HEW lysozyme
PDBid 1H6M
45
Figure 11-23 © 2017 John Wiley & Sons, Inc. All rights reserved
Outcome 2 – Inhibitor used to verify covalent lysozyme
intermediate

© 2017 John Wiley & Sons, Inc. All rights reserved 46

Discussion
• Explain the importance of conformation in the D ring of a
lysozyme substrate.

• Why was an oxonium ion expected to be involved in the lysozyme

reaction?

• Describe the experimental evidence that supports lysozyme’s

catalysis by acid-base catalysis, and transition state stabilization.
What Happens Next?

•Next week we will learn about the mechanism of serine

proteases and reaction kinetics.
•Ensure that you engage with your week 4 myLMS content
before your week 4 lecturer led session.