Enzyme Summary

ENZYMES
Learning Objectives
Explain the mode of action of enzymes in terms of an:

active site
enzyme-substrate complex
lowering of activation energy, and
enzyme specificity.


Learning Objectives
2. Follow the time course of an enzyme-catalysed reaction, by measuring :

rate of formation of products (for example using catalase), or
rate of disappearance of substrate (for example using amylase).

Learning Objectives
Investigate and explain the following effects on the rate of enzyme-catalysed reactions.
temperature
pH
enzyme concentration, and
substrate concentration
Learning Objectives
Explain the effects of
- competitive and
- non-competitive inhibitors
(including allosteric inhibitors)
on the rate of enzyme activity.
What are Enzymes? – page 2
What are Enzymes? – page 2
MODE OF ACTION FOR ENZYMES
2.1 Structure of Enzymes
Enzymes are globular proteins.
Mode of action of enzymes – page 3
ENZYMES CAN CATALYSE THE FOLLOWING REACTIONS:

Formation of 1 product by forming a new bond between 2 substrates.
Anabolism – synthesis of molecules and usually require energy. Often involve condensation reactions.

Formation of 2 products by breaking a bond in a particular substrate
Catabolism – Breakdown of molecules and usually release energy. They often involve hydrolytic reactions.
Why are enzymes so specific?
Lock-and-Key Hypothesis

Induced Fit Hypothesis

Which of the above hypothesis suggests that the substrate and the active site are NOT complementary in shape?
Enzymes carry out specific reactions
Lock-and-Key Hypothesis
Conformation of active site is complementary to that of substrate.

Enzymes carry out specific reactions
Induced Fit Hypothesis
Initial conformation of the active site might not be complementary in shape to the substrate
When substrate comes into contact with the active site, the substrate induces a conformational change in the shape of the enzyme
Structure of Enzymes – pg 5
Structure of Enzymes – pg 5
Structure of Enzymes – Active Site
Structure of Enzymes – Active Site
Structure of Enzymes – Active Site (Contact Residues)
Structure of Enzymes – Active Site (Contact Residues)
Structure of Enzymes – Active Site (Catalytic Residues)
Structure of Enzymes – globular shape maintained by other residues
How does enzymes increase the rate of reactions? – page 6
By lowering activation energy (Ea)
Activation Energy is the energy that must be overcome in order for a chemical reaction to occur.
Enzymes help to lower the Ea of the reaction  energy barrier more easily overcome

Enzymes increase the rate of reactions by lowering Ea – page 6
Mechanisms used to lower Ea (pg 7)
Enzymes help to maintain precise orientation of 2 (or more) substrates

distort/stress the bonds

Increase reactivity of substrates

Active site provide microenvironment for reaction

Active site directly participates in the chemical reaction.
Mechanisms used to lower Ea (pg 7)
Enzymes help to maintain precise orientation of 2 (or more) substrates at the active site so that reaction can occur
Mechanisms used to lower Ea (pg 7)
2. Inducing a stress in bonds of substrate.
R groups of amino acids at active site are in close promixity to certain bonds of substrate
chemical interaction between R groups and substrate  distort/stress the bonds  lower Ea to break bond
Mechanisms used to lower Ea (pg 7)
Increase reactivity of substrates
Interaction between R groups of amino acids of the enzyme and substrate can cause changes within substrate that increases its reactivity
E.g. charge on substrate is changed.
Mechanisms used to lower Ea (pg 7)
Active site provide microenvironemnt for reaction
E.g. active site with acidic amino acids may transfer the H+ to the substrate

5. Active site directly participates in the chemical reaction.
May involve brief covalent bonding with the substrate
Bond will be broken to restore active site in subsequent steps.
Mechanisms used to lower Ea (pg 7)
Enzymes help to maintain precise orientation of 2 (or more) substrates

distort/stress the bonds

Increase reactivity of substrates

Active site provide microenvironment for reaction

Active site directly participates in the chemical reaction.
QUICK CHECK (page 8)
QUICK CHECK (page 8)
ENZYMES
FACTORS AFFECTING RATE OF ENZYMATIC REACTIONS
Learning Objectives
Investigate and explain the following effects on the rate of enzyme-catalysed reactions.
temperature
pH
enzyme concentration, and
substrate concentration
Rate of rxn is affected by temp – (Pg 10)
Effects of Varying Temperature
Effects of Varying Temperature
Rate of rxn is affected by temp – (Pg 10)
Rate of rxn is affected by temp – (Pg 10)
Rate of rxn is affected by temp – (Pg 10)
5.1 Temperature affecting Rate of reaction
QUICK CHECK (page 15)

Learning Objectives
Investigate and explain the following effects on the rate of enzyme-catalysed reactions.
temperature
pH
enzyme concentration, and
substrate concentration
pH affecting Rate of reaction- enz work at max rate at optimum pH
pH affecting Rate of reaction- enz work at max rate at optimum pH
At the optimum pH, do you think the bonds are broken?
No, the intramolecular bonds, which maintain the secondary and tertiary structures of the enzyme are intact

What would the conformation of the active site be like?
the conformation of the active site is most ideal for binding of substrate.
pH affecting Rate of reaction- enz work at max rate at optimum pH
What is the frequency of successful collisions between enzyme and substrate molecules at the optimum pH?
the frequency would be the highest.

How fast would enzyme-substrate complexes will be formed?
pH affecting Rate of reaction- when pH deviates from the optimum pH
At pH lower or higher than the optimum, the concentration of hydrogen ions (H+) would have changed.

pH affecting Rate of reaction- when pH deviates from the optimum pH
5.2 pH affecting Rate of reaction- when pH deviates from the optimum pH
What happens when the charges of the R groups change?
Ionic bonds and Hydrogen bonds formed between the R groups will be disrupted.
These bonds help to maintain the conformation of the enzyme molecule.

What will happen to the active site when these bonds are broken?


5.2 pH affecting Rate of reaction- when pH deviates from the optimum pH
The conformation of the active site would be disrupted and the binding of substrate would be affected.

Rate of enzyme-catalysed reactions will decrease, as pH deviates from the optimal pH.

Therefore, enzymes work within a narrow range of pH

pH affecting Rate of reaction
pH affecting Rate of reaction
If the pH is altered by a small extent from the optimum pH, are effects normally reversible?

Yes. If the pH is restored to the optimum, the maximum activity of the enzyme will be restored.


pH affecting Rate of reaction
If the pH is altered by a small extent from the optimum pH, are effects normally reversible?

Yes. If the pH is restored to the optimum, the maximum activity of the enzyme will be restored.


Effect of pH change
What happens to the conformation of an enzyme when pH is altered to a great extent? Will it be reversible?

the conformation of the enzyme molecule would be severely affected. Denaturation of the enzyme might be irreversible.


Learning Objectives
Investigate and explain the following effects on the rate of enzyme-catalysed reactions.
temperature
pH
substrate concentration
enzyme concentration

Substrate concentration affecting rate of reaction – page 13
For a fixed enzyme concentration, the rate of reaction increases with increasing substrate concentration.
Substrate concentration affecting rate of reaction
An increase in the number of substrate molecules will result in an increase in the frequency of effective collisions between enzyme and substrate molecules.
Substrate concentration affecting rate of reaction
When the enzyme concentration is fixed, and the substrate concentration is increased,
 More enzyme-substrate complexes will be formed.
The rate of reaction will increase (till the saturation point is reached)

Substrate concentration affecting rate of reaction
Rate of reaction will reach a maximum when the point of saturation is reached, whereby increasing concentration of substrates will not increase the rate of reaction any further.
At the saturation point…
fixed number of available active sites (as the enzyme concentration remains unchanged)

the active sites of the enzyme molecules at any given moment are virtually saturated with substrate.

Max no. of enzyme-substrate formed per unit time

The enzyme/substrate complex has to dissociate before the active sites are free to accommodate more substrate.
Learning Objectives
Investigate and explain the following effects on the rate of enzyme-catalysed reactions.
temperature
pH
substrate concentration
enzyme concentration

Enzyme concentration affecting rate of reaction
In the presence of a large concentration of substrates, while pH and temperature are kept constant;

When enzyme concentration increases
Frequency of successful collisions between enzyme and substrate molecules increases
no. of enzyme - substrate complexes forms increases
Rate of reaction increases
Enzyme concentration affecting rate of reaction
CHECKPOINT
Q: At low temperatures, what happens to enzymes?
They are inactivated.

Q: Why?
Both enzymes and substrates do not have sufficient energy to have effective collisions with one another.
CHECKPOINT
CHECKPOINT
Q: What kind of bonds are easily broken when enzymes are exposed to heat?
Hydrogen bonds, ionic bonds and hydrophobic interactions
Affects structure of active site  affects formation of E-S complex

Q: What changes occur in the enzyme when pH is changed?
The charges of R groups of amino acid residues are altered.
CHECKPOINT
Q: What kind of bonds are affected?
Ionic bonds and Hydrogen bonds

Q: What will happen to the enzyme?
The conformation at the active site will change and be unable to bind substrates.
CHECKPOINT
Q: In the presence of a fixed amount of enzyme, an increase in substrate amts would lead to an increase of ________?

Frequency of effective substrate enzyme collisions.

CHECKPOINT

Q: Why does the rate of reaction reaches a max. in the presence of high [S]?

Saturation Point is reached. All the active sites on the enzymes are fully occupied. Rate of reaction is limited by the number of active sites available.
QUICK CHECK (page 15)
ENZYMES
Rate of rxn is affected by temp – (Pg 10)
pH affecting Rate of reaction
Substrate concentration affecting rate of reaction – page 13
For a fixed enzyme concentration, the rate of reaction increases with increasing substrate concentration.
Enzyme concentration affecting rate of reaction
Inhibition of enzyme reaction
Enzyme Inhibitors
Inhibition of enzymatic reactions
An inhibitor is a substance that prevents an enzyme from catalysing its reaction.

It decreases the rate of an enzyme-catalysed reaction.

The inhibitor combines with the enzyme to form an enzyme-inhibitor complex  Enzyme cannot combine with the substrate molecule.

Inhibitors can be divided into two main groups: Competitive inhibitors and non-competitive inhibitors.

Competitive Inhibition
Competitive Inhibition
5.1 Competitive Inhibition
5.1 Competitive Inhibition
Competitive Inhibition
Can the reaction still reach its maximum rate if a lot of substrate is added?
At very high substrate concentrations, the rate of reaction can reach its maximum value.
Inhibitors has no chance of binding to the active site

Competitive Inhibition
Competitive inhibition
Non-Competitive Inhibition
Non-Competitive Inhibition
Non-Competitive Inhibition
Non-Competitive Inhibition decreases the rate of reaction
Overcoming non-competitive inhibition
Non-competitive inhibition cannot be overcome even when substrate concentration increases.

The rate of reaction is unable to achieve the theoretical Vmax, unlike with competitive inhibition.
Comparing Competitive and Non-Competitive Inhibition







Checkpoint
What does a competitive inhibitor compete for?
Active site of enzyme

How can its inhibitory effect be decreased?
Add more substrates.

Can the maximum rate of reaction be reached if substrate concentration is high?
Yes.
Allosteric Enzymes
7.1 What are Allosteric Enzymes?
Allosteric inhibition is an example of reversible non-competitive inhibition.

Allosteric inhibitors bind at a site away from the active site  allosteric sites

Leads to changes in the overall structure of the enzyme and of the active site

Substrate is now unable to bind to active site of the enzyme.

Decreases rate of reaction
7.3 Mechanism of Allosteric Inhibition
When the allosteric inhibitor is not bound to the allosteric site of an enzyme, the active site of the enzyme is able to bind the substrate and catalyse the formation of product(s)
7.3 Mechanism of Allosteric Inhibition
When the allosteric inhibitor binds to the enzyme at the allosteric site, the active site of the enzyme is altered and no substrate can bind to it.
End Product Inhibition
End Product Inhibition
Enzymes whose activities are regulated by allosteric inhibitors tend to catalyse the first reaction in a biochemical pathway.

The end-product of the pathway can act as an allosteric inhibitor of the first enzyme of the pathway, thereby stopping the synthesis of the end-product.
Mechanism of Allosteric Inhibition: End Product Inhibition
A metabolic pathway usually involves a series of reactions in which each is catalysed by an enzyme.

When the end product of a metabolic pathway begins to accumulate, it may act as an inhibitor, usually on the enzyme catalysing the first reaction in the pathway


7.3 Mechanism of Allosteric Inhibition: End Product Inhibition
7.3 Mechanism of Allosteric Inhibition: End Product Inhibition
Difference between Allosteric Inhibition and Non-Competitive Inhibition
For both, the inhibitor bind to a site away from the active site


For non-competitive inhibitor (generally)  substrate may or may not still bind to active site  no catalysis

For allosteric inhibition  substrate CANNOT bind to active site (conformation is changed)  no catalysis
Co-factors in enzymatic reactions
What are enzyme Co-Factors?
Non-protein components that are required by enzymes for their activities.
May vary from simple inorganic ions to complex organic molecules.
8. Cofactors
8. Cofactors
8. Cofactors
http://www.clunet.edu/BioDev/omm/catalase/cat1.htm
8. Cofactors
8.3 Coenzymes
Coenzymes are derived from vitamins.

Eg. Nicotinamide Adenine Dinucleotide (NAD+) is an important coenzyme in respiration. It is derived from the vitamin nicotinic acid.

8.3 NAD+ as coenzyme in respiration
8.3 NAD+ as coenzyme in respiration