1.

Structure and function of enzymes

Notes
• Globular proteins acting as the body’s catalysts
• Speed up time for reaction to reach equilibrium
• Lower the activation energy of a reaction

Example:
O HO H
O LDH O
C C
NADH + H3C C H3C C + NAD+
OH OH
Pyruvic acid Lactic acid

LDH = Lactate dehydrogenase (enzyme)

NADH = Nicotinamide adenosine dinucleotide (reducing agent & cofactor)
Pyruvic acid = Substrate

© Oxford University Press, 2013

 Enzyme Structure
• All known enzymes are proteins.
• They are high molecular weight compounds made up
principally of chains of amino acids linked together by
peptide bonds.
• Many enzymes require the presence of cofactors
• Coenzyme= a non-protein organic substance which is
loosely attached to the protein part.(NAD+, NADH,
FAD)
• Prosthetic group = an organic substance which is firmly
attached to the protein or apoenzyme portion or
• Metal-ion activators ( K+, Fe++, Fe+++, Cu++, Co++,
Zn++, Mn++, Mg++, Ca++)

© Oxford University Press, 2013

The structure of an ENZYME CONTAINS
• AN Apoenzyme: Protein part
• Cofactor: Non-protein component
– Coenzyme: Organic cofactor
• Holoenzyme: Apoenzyme plus cofactor

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1. Structure and function of enzymes
Lowering the activation energy of reaction

Energy Energy
Transition state New
transition
state

Act. Act.
energy energy

Starting Starting
∆G material ∆G
material

Product Product

WITHOUT ENZYME WITH ENZYME

Note
Enzymes lower the activation energy of a reaction but DG remains the same

© Oxford University Press, 2013

1. Structure and function of enzymes
Methods of enzyme catalysis

• Provides a reaction surface (the active site)

• Provides a suitable environment (hydrophobic)

• Brings reactants together

• Positions reactants correctly for reaction

• Weakens bonds in the reactants

• Provides acid / base catalysis

• Provides nucleophilic groups

• Stabilises the transition state with intermolecular bonds

© Oxford University Press, 2013

2. The Active Site

• Hydrophobic hollow or cleft on the enzyme surface

• Accepts reactants (substrates and cofactors)

• Contains amino acids which

- bind reactants (substrates and cofactors)
- participate in the enzyme-catalysed reaction

Active site
Active site

ENZYME

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3. Substrate Binding
3.1 Induced fit

Substrate
S

Induced fit

Notes:
• Active site is nearly the correct shape for the substrate
• Binding alters the shape of the enzyme (induced fit)
• The binding process can strain bonds in the substrate
• Binding involves intermolecular bonds between functional
groups in the substrate and functional groups in the active site
© Oxford University Press, 2013
3. Substrate Binding
3.2 Bonding forces
• Ionic
• H-bonding
• van der Waals

Example vdw
interaction

S
H-bond
Active site
H ionic Phe
Ser O
bond
CO2

Asp

Enzyme

© Oxford University Press, 2013

3. Substrate Binding
3.2 Bonding forces
• Ionic
• H-bonding
• van der Waals

Example - Binding of pyruvic acid in LDH

O O
H-Bond H
C O
H3C C O

C O
O H3C C +
H3N
O
Possible interactions vdw-interactions Ionic bond

H-Bond
van der Waals
Ionic
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3. Substrate Binding
3.2 Bonding forces
• Induced fit - Active site alters shape to maximise intermolecular
bonding

S S
Ser O H Phe
H Phe
Ser O
CO2
CO2 Induced
Asp fit Asp

Intermolecular bonds not optimum Intermolecular bond lengths optimised

length for maximum bonding Susceptible bonds in substrate strained
Susceptible bonds in substrate more easily
broken
© Oxford University Press, 2013
3. Substrate Binding
Example - Binding of pyruvic acid in LDH

O
H

C O
H3C C
+
H3N
O

© Oxford University Press, 2013

3. Substrate Binding
Example - Binding of pyruvic acid in LDH

O
p bond H
weakened
O

C O
H3C C
+
H3N
O

© Oxford University Press, 2013

4. Catalysis mechanisms
4.1 Acid/base catalysis
• Histidine
+H+
NH NH
-H+
N N
H
Non-ionised Ionised
Acts as a basic catalyst Acts as an acid catalyst
(proton 'sink') (proton source)

4.2 Nucleophilic residues

H H
H3N CO2 H3N CO2

L-Serine L-Cysteine
OH SH

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4. Catalysis mechanisms
Serine acting as a nucleophile

Substrate

Product
HO
H2O
O OH
OH

Ser Ser Ser

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4. Catalysis mechanisms
Mechanism for chymotrypsin

Catalytic triad of serine, histidine and aspartate

:N H
.. N
:O H O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

: O:
C
Protein NH Protein

:N H
.. N
:O H O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

:
:O:
Protein C NH Protein

:N H
N
:O H O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

:
:O:
Protein C NH Protein

+ H
H N N
:O: O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

H H
O :

:
:N H
N
: : O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

:
:O: H

Protein O
C H

:N H
N
:O : O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

:
:O: H

Protein O:

:
C

H H
N N
:O : : O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin

:O:
C
Protein OH

:N H
.. N
:O H O O

Ser His Asp

Chymotrypsin

© Oxford University Press, 2013

4. Catalysis mechanisms
Mechanism for chymotrypsin
:O:
C
protein NH protein

.. :N N H
:O H O O

Ser His Asp

.. ..
:O : :O :
H H
C C prote in
O

:
O

:
protein NH protein protein NH protein

:
:
C
H
:N N H N H :N N H
:O H O O :O : N
O O :O : O O

Ser His Asp Ser His Asp Ser His Asp

.. H .. protein OH
:O: : O : .. C
protein O
.. H protein OH
.. O
C C
:N N H
H
N H .. :N N H
:O : O O :O : N
O O : OH O O

Ser His Asp Ser His Asp Ser His Asp

© Oxford University Press, 2013
5. Overall Process of Enzyme Catalysis

S P
S P

EE E E E

E+S ES EP E+P

• Binding interactions must be strong enough to hold the substrate

sufficiently long for the reaction to occur
• Interactions must be weak enough to allow the product to depart
• Interactions stabilise the transition state
• Designing molecules with stronger binding interactions results in
enzyme inhibitors which block the active site

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6. Regulation of Enzymes
• Many enzymes are regulated by agents within the cell
• Regulation may enhance or inhibit the enzyme
• The products of some enzyme-catalysed reactions may act as inhibitors
• Usually bind to a binding site called an allosteric binding site

NH2

Example N
N

O N N
O P O
O AMP
O H H
H H
OH OH

H OH
H OH
H O
H O
HO H
Glycogen O HO
H H OH O
HO
H OH Phosphorylase a H O P
H OH
HO OH n
Glucose-1-phosphate

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6. Regulation of Enzymes
Active site
Active site
unrecognisable

Induced
ACTIVE SITE
fit
(open)
Enzyme
ENZYME
(open)
Enzyme
ENZYME
Allosteric
binding site

Allosteric
inhibitor

• Inhibitor binds reversibly to an allosteric binding site

• Intermolecular bonds are formed
• Induced fit alters the shape of the enzyme
• Active site is distorted and is not recognised by the substrate
• Increasing substrate concentration does not reverse inhibition
• Inhibitor differs in structure to the substrate © Oxford University Press, 2013
6. Regulation of Enzymes

Biosynthetic pathway

S P P’ P’’ P’’’

Enzyme
(open)
ENZYME

Inhibition Feedback control

Notes
• Enzymes with allosteric sites are often at the start of a biosynthetic pathway
• The enzyme is controlled by the final product of the pathway
• The final product binds to the allosteric site and switches off the enzyme

© Oxford University Press, 2013

6. Regulation of Enzymes
• External signals can regulate the activity of enzymes
(e.g. neurotransmitters or hormones)
• Chemical messenger initiates a signal cascade which activates
enzymes called protein kinases
• Protein kinases phosphorylate target enzymes to affect activity

Example

Phosphorylase b
(inactive) Signal
Protein cascade
kinase Adrenaline
Phosphorylase a
(active)

Glycogen Glucose-1-phosphate
Cell
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