CELLULAR BIOCHEMISTRY

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CELLULAR BIOCHEMISTRY PROTEINS AND ENZYMES -
LECTURE 8 ENZYMES - MECHANISM OF ACTION -
LYSOZYME
FACTORS THAT ENHANCE REACTION RATE LYSOZYME
MECHANISM
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MECHANISMS OF ENZYME ACTION - GENERAL ASPECTS
FACTORS RESPONSIBLE FOR ENZYMIC CATALYSIS 1.
Proximity and orientation of substrate 2.
Formation of specific, unstable covalent
intermediates 3. Acid-base catalysis 4.
Strain and stabilisation of transition state
intermediates 5. Induced fit 6. Hydrophobic
environment For any given enzyme several of the
above factors will apply and the enhanced
reaction rate is therefore the product of several
separate enhancements. Further reading Stryer
5th edn. Chapters 8 and 9. For lysozyme
mechanism of action - Stryer 4th Edn, pp.207-216
gives a full and clear account
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• Proximity and orientation of substrate
• Binding of two or more substrates together at the
  active site increases their effective
  concentrations.
• Specific binding at the active site can orientate
  the substrates precisely with regard to each
  other and the catalytic groups on the enzyme.
  Koshland has suggested that such orientation may
  involve orbital steering, the electron shells
  being brought into very precise proximity. Note
  that these factors contribute to specificity as
  well as catalysis.

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Formation of specific, unstable covalent
intermediates
Chymotrypsin, acting as an esterase, cleaves
p-nitrophenyl acetate to give a yellow product,
p-nitrophenol. The reaction goes in two
stages chymotrypsin p-nitrophenyl acetate
gt acetyl chymotrypsin p-nitrophenol
fast acetyl
chymotrypsin water gt chymotrypsin
acetate slow By lowering
the pH rapidly, the acetyl chymotrypsin
intermediate can be isolated. The acetyl is
attached covalently to a serine side-chain at the
active site.
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Acid-base catalysis
In the chemistry laboratory acid-base catalysis
is achieved at extremes of pH. Enzymes achieve
general acid-base catalysis at near neutral pH by
using amino acid side chains that can act as as
proton donors (acids) or proton acceptors
(bases). For example in lysozyme a glutamic
acid side chain -(CH2)2-COOH donates a proton
to the susceptible bond in the polysaccharide
substrate, causing cleavage. Two side chain
groups that can act as proton donors Two
side chain groups that can act as proton
acceptors
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• Strain and stabilisation of transition state
  intermediates
• Often when the substrate binds to its active
  site, part of its conformation is forced to
  change in order for it to 'fit. The new
  conformation favours formation of transition
  state intermediates (i.e. such intermediates are
  stabilised by the geometry of the active site)
  and destabilises the substrate.
• The reaction is therefore facilitated. Note
  that the strain imparted to the conformation of
  the substrate is a consequence of good binding of
  the enzyme to the rest of the substrate (high
  affinity).
• Again lysozyme is a good example as the
  polysaccharide substrate binds to the active site
  it mostly fits well (facilitating binding) but
  the sugar residue next to the cleavable bond is
  distorted, destabilizing the substrate, but
  stabilizing the transition state intermediate.

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Induced fit Koshland's theory. The conformation
of the enzyme may change as the substrate binds,
contributing to the overall strain in the E-S
complex. Also, induced fit may play an
important role in specificity as well as
catalytic activity. It can explain why an enzyme
like hexokinase can phosphorylate sugars, but not
H2O. Hexokinase catalyses glucose ATP
gt glucose-6-phosphate ADP but not H2O
ATP gt phosphate ADP (the second
reaction would waste ATP, and has to be
avoided) Induced fit here means that the
enzyme's conformation changes (and the active
site becomes active) only when glucose is bound
to the active site. With just water in the
active site, the conformation is the inactive
one, and the water cannot be phosphorylated.
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CONFORMATIONAL CHANGE OF HEXOKINASE
(From Stryer Ch 16)
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• Hydrophobic environment
• Water is often a poor solvent in which to carry
  out chemical reactions because the reactive
  groups become solvated (hydrated) and surrounded
  by a shell of water molecules that prevents the
  approach of another reactant.
• Organic chemists usually carry out their
  reactions in organic solvents which are less
  effective in shielding groups in this way.
• The active site of an enzyme often contains a
  number of hydrophobic side chains and, as the
  substrate binds, water molecules are squeezed out
  and a hydrophobic micro-environment is
  established in which hydration/shielding of
  reactive groups is minimised.
• So the environment in the immediate region of the
  reactive groups resembles that favoured by the
  organic chemist.

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LYSOZYME SUBSTRATE

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Lysozyme
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Lysozyme NAG3
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LYSOZYME INHIBITOR

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LYSOZYME ACTION WHICH BOND IS CLEAVED?

1. Rate of hydrolysis of oligomers of NAG
(relative) (NAG)2 0
(NAG)3 1
(NAG)4 8 (NAG)5
4,000 (NAG)6 30,000
(NAG)8 30,000 2. (NAG)3
is stable, so probably A-B and B-C bonds are not
cleaved 3. Site C on enzyme cannot be filled by
NAM (lactyl side-chain will not fit), so
bacterial substrate must fit
NAG-NAM-NAG-NAM-NAG-NAM A B
C D E F Lysozyme
specifically cleaves a NAM-NAG bond, so C-D and
E-F cannot be cleavage sites. By elimination
cleavage site must be between D and E
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LYSOZYME ACTION WHICH SIDE OF D-E BOND IS CLEAVED?

Distinguished by use of water enriched with 18O
Skeleton structures only. So, cleavage must be
at position a
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MECHANISM OF ACTION OF LYSOZYME

Cleavage of (NAG)6 NAG-NAG-NAG-NAG-NAG-NAG
A B
C D E F 1. -COOH of Glu-35
donates H to bond between C1 of D ring and
glycosidic oxygen, cleaving bond
2. This creates a positive charge on C1 of D NAG
residue - a transient intermediate carbonium ion.
Potentially very unstable, but stabilised by
distortion of D-ring and proximity of negative
charge on Asp-52.
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MECHANISM OF ACTION OF LYSOZYME (CONT)

• Cleavage of (NAG)6 NAG-NAG-NAG-NAG-NAG-NAG
• A B
  C D E F
• 3. Dimer (residues E-F) diffuses away
• 4. Carbonium ion intermediate reacts with OH-
  from solvent

5. (NAG)4 diffuses away. Glu-35 is reprotonated.
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MECHANISM OF ACTION OF LYSOZYME (CONT) CHAIR AND
HALF-CHAIR CONFORMATIONS

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MECHANISM OF ACTION OF LYSOZYME (CONT) pH PROFILE

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MECHANISM OF ACTION OF LYSOZYME (CONT) TRANSITION
STATE ANALOGUE

The lactone analogue resembles the
transition-state intermediate because its D ring
has a half-chair form
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MECHANISM OF ACTION OF LYSOZYME

Contributions of each sugar of (NAG)6 to standard
free energy of binding of substrate