Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 4200
II. Macromolecular Interactions
Structure and Mechanism II
Lysozyme
Lysozyme
Biological function:
Facilitates changes in the cell wall during growth and division
In eucaryotes, its found in bodily secretions (tears, egg whites, etc)
→ part of the host defensive system
Lysozyme hydrolyzes polysaccarid chains and ruptures certain bacterial
cells by breaking down of the cell wall.
1
Bacterial Cell Walls
The cell walls of both gram-positive and
Gram negative bacteria consist of covalently
linked polysaccharide and polypeptide chains
Which form a baglike molecule that
completely encases the cell
Gram-positive bacteria
This framework is known as peptidoglycan
or murein. Linear chains of alternating
β(1→4)-linked N-acetylglucoseamine (NAG)
and N-acetylmuramic acid (NAM).
NAM forms an amid bond with a D-amino acid
containig tetrapeptide to form the
peptidoglycan repting unit
Gram-negative bacteria
Bacterial Cell Walls
This framework is known as peptidoglycan
or murein. Linear chains of alternating
β(1→4)-linked N-acetylglucoseamine (NAG)
and N-acetylmuramic acid (NAM).
NAM forms an amid bond with a D-amino acid
containig tetrapeptide to form the
peptidoglycan repting unit
2
Lysozyme Reaction
Lysozyme hydrolyses the glycosidic bond of the (NAM-NAG)n heteropolymer that is the backbone of the bacterial cell wall.
Specific for NAM-NAG β(1→4) glycosidic bonds
It also hydrolyses β(1→4) poly N-acetylglucosamine (NAG).
What’s that?
Homopolysaccharides
Chitin: linear homopolysacharid composed
of β(1→4) N-acetylglucosamine
Linear; indigestible by mammals
Serves structural role in anthropods
Is a cell wall component of most
fungi.
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Hen Egg White Lysozyme
Hen egg white (HEW) lysozyme is the most widely studied species of
lysozyme and is one of the mechanistically best understood enzymes.
But the actual mechanism is
still subject of controversy.
Small protein of
129 amino acid residues
14.7 kD
4 internal disulfide bridges
And you even know how to purify it!
HEW Lysozyme Reaction
Mechanism of the nonenzymatic
acid-catalyzed hydrolysis of an
acetal to a hemiacetal.
4
Understanding the Mechanism of Lysozyme
Action
Binding Studies → The binding site:
Compound
kcat
(NAG)2
(NAG)3
(NAG)4
(NAG)5
(NAG)6
(NAG-NAM)3
2.5 x 10-8
8.3 x 10-6
6.6 x 10-5
0.033
0.25
0.5
Understanding the Mechanism of Lysozyme
Action
X-Ray structure of Lysozyme:
Lysozyme’s catalytic site was
identified by model building.
(Philipps 1972)
6 discrete binding sites have been
identified (A-F)
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Understanding the Mechanism of Lysozyme
Action
Binding Studies → The binding energies:
Compound
NAG
NAM
NAG
NAM
NAG
NAM
Site
A
B
C
D
E
F
kJ·mol-1
- 7.5
- 12.3
- 23.8
+12.1
- 7.5
- 7.5
Why is the binding energy for D so high?
Understanding the Mechanism of Lysozyme
Action
pH vs. rate
Between pH 4-6, lysozyme
is optimal active.
What does that suggest?
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Understanding the Mechanism of Lysozyme
Action
pH vs. rate
Between pH 4-6, lysozyme
is optimal active.
- one or more ionizable groups with
a pKa of ~ 4
→ group(s) are protonated
below pH 4
- one or more ionizable groups with
a pKa of ~ 6
→ group(s) are deprotonated
above pH 6
Understanding the Mechanism of Lysozyme
Action
pH vs. rate
Between pH 4-6, lysozyme
is optimal active.
- one or more ionizable groups with
a pKa of ~ 4
→ group(s) are protonated
below pH 4
- one or more ionizable groups with
a pKa of ~ 6
→ group(s) are deprotonated
above pH 6
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Understanding the Mechanism of Lysozyme
Action
Amino acids with pKa’s near 4
include Asp and Glu
Amino acids with pKa’s near 6
include His.
Hypothesis: Glu/Asp and His residues are the
catalytic residues.
But be careful ionizable groups that participate in
catalysis often have abnormal (perturbed) pKa’s
Understanding the Mechanism of Lysozyme
Action
Amino acids with pKa’s near 4
include Asp and Glu
Amino acids with pKa’s near 6
How to test the Hypothesis
?
include
His.
Hypothesis: Glu/Asp and His residues are the
catalytic residues.
But be careful ionizable groups that participate in
catalysis often have abnormal (perturbed) pKa’s
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Understanding the Mechanism of Lysozyme
Action
Protein modification
Carbodiimides specifically react with carboxylates
Carbodiimide modification of a carboxylic group in a protein, followed by
rearrangement to yield a stable N-acylurea.
When reacted with lysozyme the enzyme is inactivated.
BUT when reacted with with lysozyme in the presence of substrate:
→ a portion of the activity is retained.
Understanding the Mechanism of Lysozyme
Action
Iodoacetamides react specifically with His at low pH.
Imidazole specific reagents have no effect on the activity
lysozyme under any conditions.
What can we conclude from that?
In conjunction with the pH dependence experiments:
→ the catalytic residues are all carboxylates
→ the pKa for one carboxylate is surprisingly high (perturbed)
→ is in an apolar environment.
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Understanding the Mechanism of Lysozyme
Action
Stereochemistry
The absolute configuration of the
N-acylmuramic acid C1 is retained
in the product.
Lysozyme is sometimes referred to as a
retaining glycosidase.
Retaining glycosidases can have one of
two mechanisms:
Which?
Stereochemistry of Lysozyme
1. disassociative or displacement mechanism
-a trigonal planar transition state intermediate is formed
-as the C1-O1 bond is broken prior to H2O addition
Problem here ther intermediate can be attacked form either face of
the ring.
→ Philipps Mechanism
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Stereochemistry of Lysozyme
2. associative or “ping-pong” mechanism
- Two consecutive SN attacks on the C1 of NAM will lead to an
overall retention of its absolute configuration.
-The transition state is a trigonal bipyramid
- Typically involves the formation of an acyl-enzyme intermediate
No acyl-enzyme intermediates have been isolated so far.
Understanding the Mechanism of Lysozyme
Action
X-Ray structure of Lysozyme:
Lysozyme’s catalytic site was
identified by model building.
(Philipps 1972)
6 discrete binding sites have been
identified (A-F)
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Understanding the Mechanism of Lysozyme
Action
Binding Studies → The binding energies:
Compound
Site
NAG
NAM
NAG
NAM
NAG
NAM
kJ·mol-1
A
B
C
D
E
F
- 7.5
- 12.3
- 23.8
+12.1
- 7.5
- 7.5
Why is the binding energy for D so high?
Understanding the Mechanism of Lysozyme
Action
Binding Studies → The binding energies:
Compound
Site
NAG
NAM
NAG
NAM
NAG
NAM
C
kJ·mol-1
A
B
C
D
E
F
D
- 7.5
- 12.3
- 23.8
+12.1
- 7.5
- 7.5
E
F
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Stereochemistry of Lysozyme
1. disassociative or displacement mechanism
-a trigonal planar transition state intermediate is formed
-as the C1-O1 bond is broken prior to H2O addition
Problem here ther intermediate can be attacked form either face of
the ring.
Lets formulate a mechanism!
The Philips Mechanism
1. Lysozyme attaches to a bacterial
cell watt by binding to the
hexasacharid unit.
→ distorting residue D towards the
half-chair conformation
2. Glu53 transfers proton to O1 of the
D ring (general acid catalysis).
→C1-O1 bond is cleaved, generating
a resonance-stabilized oxonium
ion on C1.
3. The ionized Asp52 stabilizes the
developing charge on the oxonium
ion (charge-charge interaction)
→electrostatic catalysis
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The Philips Mechanism
4. Ring E is released (leaving group), yielding a cationic
noncovalent glycosyl-enzyme intermediate.
→ Water is added from the solution in a reversal
of the preceding steps.
→ Retention of the configuration is achieved by
shielding of one of the oxonium ion’s faces.
Testing the Phillips Mechanism
Identification of the catalytic residues
Important catalytic groups have been experimentally
identified through site-directed mutagenesis.
Glu53Gln → catalytic activity < 0.1 % of WT
→ binding only ~1.5 fold decreased
→ essential for catalysis
Asp52Asn → catalytic activity ~ 5% of WT
→ binding ~2 fold increased
→ essential for catalysis
Other carboxyl groups are not involved (chem. modification)
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Testing the Phillips Mechanism
The Role of Strain
The residue in position D is distorted towards
the half-chair conformation in response to
the unfavorable contacts (sterical hindrance)
Inhibitor studies:
NAG-lactone is an excellent inhibitor of
lysozyme.
It is a transition state analog that has half-chair conformation
→ supports the oxonium ion transition state.
But ?
NAG-Lacton as Transition State Analog
Planarity at C1 also
required for associative
mechanism
→ trigonal bipyramidal
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NAG-Lacton as Transition State Analog
The role of substrate distortion can be questioned:
Binding affinities of various substrate analogs for the S site have
been determined:
The affinity for NAG lactone is only 9.2 kJ/mol higher than for NAG
→ ~ 40 fold rate enhancement as result of strain!
(rate enhancement by lysozym ~108 fold)
NAG-Lacton as Transition State Analog
The role of substrate distortion can be questioned:
N-acetylxylosamine (XylNac) has only a marginal greater
binding affinity for the D site (-3.8 kJ/mol) than NAG (-2.5 kJ/mol)
The Philipps mechanism postulates that sterical clashes between
the C6-group and the protein lead to the half-chair conformation.
Still the best argument for the Phillips mechansim is the absence of a
detectible acyl-enzyme intermediate.
And as always….
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X-ray Structure of a HEW Lysozyme Covalent
Intermediate
Disassotiaticve oxo-carbon
mechanism
Associative acyl-enzyme
mechanism
What can we do?
X-ray Structure of a HEW Lysozyme Covalent
Intermediate
Associative acyl-enzyme
mechanism
The reason why no acyl-enzyme
intermediate was isolated
rate of breakdown
to fast
What can we do?
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X-ray Structure of a HEW Lysozyme Covalent
Intermediate
Associative acyl-enzyme
mechanism
Formation of oxonium ion is
slowed down by electron
withdrawal using
NAG2FGlcF
slowed down
Mutating Glu35 to Gln removes
the general acid-base catalysis
stabilizes
Use of a substrate with better leaving group (F)
X-ray Structure of a HEW Lysozyme Covalent
Intermediate
ESI-MS analysis of HEW Lysozyme complexes
a) WT HEW Lysozyme
b) E35Q HEW Lysozyme with NAG2F
c) WT HEW Lysozyme with NAG2FGlcF
d) E35Q HEW Lysozyme with NAG2FGlcF
PDBid 1H6M
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X-ray Structure of a HEW Lysozyme Covalent
Intermediate
Phillips mechanism or not?
X-ray Structure of a HEW Lysozyme Covalent
Intermediate
The Phillips mechanism must be altered to take into account
the transient formation of a covalent glycosyl-enzyme ester
intermediate.
The formation of a long-lived ion pair is unlikely.
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Schematic Diagram of the Typical Layout of an
Electrospray Source
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