Enzymes

Question 1

(p) Explain the mode of action of enzymes in terms of an active site, enzyme-substrate complex, lowering of activation energy and enzyme specificity using the lock-and-key and induced-fit hypotheses.

Answer 1

Active Site
- Enzymes have a unique three dimensional
conformation with an active site
- The active site is formed by 3 to 12 amino acids from
different parts of a single polypeptide chain held
together by H^2ID.

Enzyme specificity
- The enzyme specificity is determined by the fit
between the shape of the enzyme’s active site and its
substrate.
- The active site of an enzyme is complementary
to its substrate in terms of shape, size, charge and
orientation.
- The enzyme specificity is a result of its three
dimensional conformation, which is consequence of
its amino acid sequence.

Enzyme-Substrate Complex
- When the substrate binds to the active site of the
enzyme, the enzyme-substrate complex (E-S complex)
is formed.
- The substrates are held in active site by weak bonds
such as hydrogen bonds, ionic bonds and
hydrophobic interactions.
- The R groups of a few of the amino acids that make
up the active site (catalytic site) catalyze the
conversion of substrate to product.
- Once the products are formed, they are no longer
complementary to the active site and thus, they will
leave the enzyme.

Lock-and-key hypothesis
- The enzyme acts as a lock and the substrate acts as a
key, which fits precisely.
- The active site of the enzyme is perfectly
complementary to the substrate in terms of shape,
size, charge and orientation.
- This mode of action is more probable for enzymes
that work on only one type of substrate.

Induced fit Hypothesis
- Enzymes may work in a more flexible manner.
- The active site is not perfectly complementary to the
substrate in terms of shape, size and orientation.
- However, upon forming some bonds with the
substrate, the enzyme changes its shape, which leads
to a precise fit to form the enzyme-substrate complex. - This mode of action is more probable for
enzymes that work on a group of closely-related
substrates, e.g. lipases.

Activation Energy

• Activation energy is often supplied in the form of thermal energy (heat) that the reactant molecules absorb from the surroundings.
• With increase in temperature, the reactants gain kinetic energy, thus colliding more frequently and more forcefully.
• Thermal agitation of atoms in the molecules contorts the reactants, making the bonds more likely to break.
• When the molecules have absorbed enough energy for the bonds to break, the reactants are in an unstable condition known as the transition state.
• Enzymes speed up biological reactions as they provide an alternative pathway with lower activation energy.
• They lower the Ea of the reaction by promoting the formation of the transition state by:
  1. Allowing close proximity of reactants due to temporary binding of substrates on the enzyme.
  2. Ensuring correct orientation of the reactants
  3. Destabilizing the bonds of reactants
  4. Providing a microenvironment conducive for reaction

Question 2

Investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration of an enzyme-catalyzed reaction by measuring rates of formation of products (e.g. measuring gas produced using catalase) or rate of disappearance of substrate (e.g. using amylase, starch and iodine)

Answer 2

The Michaelis constant or Km of an enzyme is the:
- Substrate concentration at which the rate of reaction
catalyzed by the enzyme equals to half its maximum
rate (i.e. ½ Vmax).

Substrate Concentration
Description:
- At low substrate concentration, there is a linear /
proportional increase in rate of reaction with increase
in substrate concentration.
- As substrate concentration continues to increase,
the increase in rate of reaction slows down.
- Any further increase in substrate concentration will
not increase the rate of reaction. Rate of reaction
plateaus off and maximum rate of reaction is
reached.

Explanation:
At low substrate concentration,
- increase in substrate concentration increases the
number of effective collisions between enzyme and
substrate molecules. Not all the active sites of the
enzyme molecules present will be occupied at any
one time.
- There is an increase in the number of enzyme-
substrate complex formed per unit time. Hence, there
is a proportional increase in the rate of formation of
products.
- Substrate concentration is limiting the rate of
reaction.
At high substrate concentration,
- further increase in substrate concentration results
in all active sites of enzymes being occupied by the
substrate at any one time.
- Maximum number of enzyme-substrate complex
formed per unit time. Hence, the rate of formation of
products has reached the maximum.
- Other factors like enzyme concentration is now
limiting the rate of reaction.

(Explanation for Enzyme conc. similar to Substrate conc.)

Temperature
Explanation:
At very low temperatures,
- substrate and enzyme molecules have low kinetic
energy.
- Thus, they move very slowly and there are very few
effective collisions between enzyme and substrate
molecules. The rate of enzyme-substrate complex
formation is thus very low.
As the temperature increases to optimum temperature,
- the kinetic energy of molecules increases and
molecules move faster. (rate doubles for every 10
degrees increase in temp.)
- Thus, there are more effective collisions between
enzyme and substrate molecules. There is an
increase in the number of enzyme-substrate
complex formed per unit time.
- Also, substrate molecules at higher energy levels
have a higher probability to overcome the activation
energy barrier and form products.
At optimum temperature,
- Maximum number of enzyme-substrate complex
formed per unit time.
At temperatures slightly above optimum temperature, - thermal agitation of enzyme molecules disrupts the
weaker bonds such as hydrophobic interactions,
hydrogen bonds, and ionic bonds.
- This distorts the specific 3-dimensional conformation
of the enzyme.
- Thus the active site is distorted and no longer
complementary to the substrate.
- As a result, substrate molecules cannot fit the active
site of enzyme molecules and E-S complex cannot be
formed. The enzymes are said to be denatured.
- With an increase in percentage of enzymes that are
denatured, the rate of E-S complex formation will
decrease.
At very high temperatures,
- there are also an increasing percentage of enzymes
that are denatured. When all enzymes are eventually
denatured, the rate of reaction then falls to zero.

pH
Explanation:
At optimum pH,
- all the ionic and hydrogen bonds between R groups
of amino acids are intact.
- The active sites are complementary to the
substrate molecule. As such, maximum number of
enzyme-substrate complex can be formed per unit
time.
A slight change in pH from optimum pH
- will change the charge found on the acidic and basic
R-groups of amino acids at the active site. - This reduces the binding ability of substrate to the
active site and hence the rate of formation of E-S
complex will decrease.
A drastic change in pH from optimum pH
- will disrupt the ionic bonds between the acidic and
basic R-groups of the amino acids and hydrogen
bonds between polar R-groups.
- This distorts the specific 3-dimensional conformation
of the enzyme.
- Thus the active site is distorted and no longer
complementary to the substrate.
- As a result, substrate molecules cannot fit the active
site of enzyme molecules and E-S complex cannot be
formed. The enzymes are said to be denatured.
- With an increase in percentage of enzymes that are
denatured, the rate of E-S complex formation will
decrease drastically. When all enzymes are
eventually denatured, the rate of reaction then falls to
zero.

Question 3

(r) Describe the structure of competitive and non-competitive inhibitors with reference to the binding sites of the inhibitor.

Answer 3

Competitive inhibition
- Competitive inhibitors are structurally similar (in terms
of shape, size, charge and orientation) to the
substrate molecule.
- bind to the active site of the enzyme and thus
competes with the substrate for the active site.
- reduce the number of active sites available for the
substrates to bind and form enzyme-substrate (E-S)
complex

Non competitive inhibition
- Non-competitive inhibitors are not structurally similar
(in terms of shape, size, charge and orientation) to the
substrate molecule.
- bind at a site away from the active site
- This interaction alters the specific 3-dimensional
conformation of the enzyme molecule such that
o active site is distorted and no longer complementary
to substrate, thus not able to bind to the substrate
properly

Question 4

(s) Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity.

Answer 4

Competitive inhibition
- Km of the enzyme will increase in the presence of
competitive inhibitor.
- Vmax can be reached eventually at higher substrate
concentration.
- When substrate concentration is low, it is more likely
for the enzyme to collide with competitive inhibitor
molecules and form enzyme-inhibitor (E-I) complex.
o Thus, the number of active sites available for
substrate molecules will be reduced and the rate of
E-S complex formation will decrease.
- When substrate concentration is high, it is more likely
for the enzyme molecules to collide with substrate
molecules and form enzyme-substrate (E-S) complex. o Thus, the competitive inhibitor has no effect on
maximum rate of reaction.

Non competitive inhibition
- Km of the enzyme remains unchanged in the
presence of non-competitive inhibitor since non-
competitive inhibitors do not compete with substrate
molecules for active site.
- Vmax is lowered as non-competitive inhibitors
reduce the number of functional enzymes. Thus
Vmax will not be restored even if substrate
concentration is increased.

Allosteric Activation and Inhibition
- Most allosterically regulated enzymes are constructed
from two or more subunits.
- In allosteric regulation, an activator or inhibitor binds
to an allosteric site, often located where subunits join. o The binding of an activator to a regulatory site
stabilizes the shape that has functional active site. o The binding of an inhibitor stabilizes the inactive
form of the enzyme.