13 Biochemistry Lecture - Enzymes 2

Faculty of
Medicine
Medical
Education-
Damietta
University
Level 1
Semester 1
Module 1B
Level 1
Semester 1
Module 1B
Introduction to Biochemistry
and
Basis of Genetics
Catalytic Proteins 2: Enzymes
Mechanism of Enzyme Action

• Contact: Medical Biochemistry Department.
• email: [email protected]
• Mobile: 01141512123
• Academic hours:
➢Sunday: 10:00-12:00 AM
➢Wednesday: 10:00-12:00 AM
Contents
• Enzyme:
➢ Mechanism of enzyme action.
➢ Factor affecting enzyme activities.
➢ Enzyme Kinetics.
Learning outcomes
At the end of the lecture, the students should be able to:
• Explain the mechanism of enzyme action
• List, identify and describe different factors affecting

enzyme activities and their effects.
• Describe and explain enzyme Kinetics.
• Correlate their knowledge to a clinical situation.

Case scenario
(Clinical correlate)
• Go in a trip of a grilled meat in our

alimentary tract, the PH of GIT parts
differ to help its digestion. How does it
work?
Catalytic Protein
(Enzyme) Chemistry
ILO-1
Explain the mechanism of enzyme action

Mechanism of enzyme action
1- The substrate (S) binds to the enzyme (E) at its active
catalytic site to form activated intermediate enzyme
substrate complex (ES).
2- The activated complex (ES) cleaved to the products (P)

and the original enzyme (E)
3- In conjugated protein enzymes: coenzyme acts as an

acceptor for one of the products helping the cleavage of
the enzyme substrate complex.
Enzymatic reaction steps
1. Substrate approaches active site

2. Enzyme-substrate complex forms
3. Substrate transformed into products
4. Products released
5. Enzyme recycled
Theories of E-S binding
Lock and Key model
Proposed by Fischer in 1894 Size
doesn’t matter…
Shape matters!
- In this model, the active sites of the unbound enzyme is

complementary (fit) in shape to the substrate
- The substrate fits in this catalytic site in a similar way to lock and
key. The key will only fits its own lock .
S
+
E-S
E
P
2- Induced fit theory: -
• It is a more flexible model, where the
catalytic site is not fully formed.
• The catalytic site of the enzyme is not
complementary to the substrate.
• Binding of the substrate to the enzyme
induces changes in the shape of the catalytic
site making it more fit for substrate.
Induced-fit model
Proposed by Koshland in 1958
In this model, the enzyme changes shape on

substrate binding
Induced fit theory:
Flexible model
Enzyme action
1- Enzymes increase the rate of reaction by

decreasing the activation energy of reaction.
2- The activation energy is needed to overcome

the energy barrier between reactants and
products.
• So what’s a cell got to do to reduce
activation energy?
– get help! … chemical help… ENZYMES
Call in the
ENZYMES!
G
Catalysts Work by Reducing the Activation
Energy of a Reaction
Pheeew…
that takes a lot
less energy!
Enzymes
Lower a
Reaction’s
Activation
Energy
Mechanism of Enzyme Activity
A substrate(s) fits into a binding site on the enzyme.
The enzyme lowers the energy required to reach the transition state.
The product no longer fits the binding site and is released.

Activity
• 1-The “lock and key” model of enzyme action
illustrates that a particular enzyme molecule
(A) forms a permanent enzyme-substrate complex
(B) may be destroyed and resynthesized several
times
(C) interacts with a specific type of substrate
molecule which is complementary to its shape
(D) reacts at identical rates under all conditions
Catalytic Protein
(Enzyme) Chemistry
ILO-2
List, identify and describe different

factors affecting enzyme activities and
their effects.
Factors Affect Rate of Enzyme
Action
• Enzyme concentration
• Substrate concentration
• Temperature
• pH
• Concentration of coenzymes
• Concentration of ion activators
• Time
• Inhibitors
Factors affecting the rate
of enzyme action
1- Effect of enzyme concentration
The rate of enzyme action is directly proportional

to the concentration of enzyme provided that
there are sufficient supply of substrate &
constant conditions.
2- Effect of substrate concentration
-The rate of reaction increases as the substrate concentration

increases up to certain point at which the reaction rate is maximal
(Vmax.)
At Vmax, the enzyme is completely saturated with the substrate any

increase in substrate concentration doesn't affect the reaction rate.
Substrate concentration
What’s
happening here?!
reaction rate
substrate concentration
Michaelis constant (Km)
- It is the substrate concentration that
produces half maximum velocity of enzyme
• Enzymes with low Km: have high affinity to the
substrate i.e. they act at maximal velocity at low
• E.g. Hexokinase acts on glucose at low concentration

(fasting state)
• Enzymes with high Km: they have low affinity to the

substrate i.e. they act at maximal velocity at high
• E.g. Glucokinase enzyme acts on glucose at high

concentration (fed state)
Concentration of
substrate
Km
3- Effects of temperature
- Rate of reaction increases gradually with

the rise in temperature until reach a
maximum at a certain temperature, called
optimum temperature
- The optimum temperature is 37-40 °C in

humans
Temperature
What’s
happening here?!
reaction rate
37°
temperature
Effect of temperature on reaction rate is due to:
1- Increase of temperature increase the initial energy of

substrate and thus decrease the activation energy
2- Increase of collision of molecules: more molecules

become in the bond forming or bond breaking
distance.
* After the optimum temperature, the rate of reaction

decrease due to denaturation of the enzyme (60-65
C).
4- Effect of PH
- Each enzyme has an optimum PH at

which its activity is maximal
• E.g. Optimum PH of pepsin = 1.5 - 2
• Optimum PH of pancreatic lipase = 7.5 - 8
• Optimum PH of salivary amylase = 6.8

pH
What’s
happening here?!
Salivary
pepsin amylase
trypsin
reaction rate
pepsin
trypsin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
Change of PH above or below optimum PH
decrease rate of enzyme action due to:
1- The enzyme activity depends on the ionization

state of both enzyme and substrate which is
affected by PH.
2- Marked change in PH will cause denaturation

of enzyme.
5- Concentration of coenzymes:
In the conjugated enzymes that need

coenzymes, the increase in the coenzyme
concentration will increase the reaction
rate.
6- Concentration of ion activators: The
increase in metal ion activator increase
the reaction rate
Enzymes are activated by ions:
1- Chloride ion activate salivary amylase
2- Calcium ion activate thromobokinase

enzyme
7- Effect of time:
• In an enzymatic reaction, the rate of

reaction is decreased by time.
• This is due to:
1- The decrease in substrate concentration.
2- The accumulation of the end products.
3- The change in PH than optimum PH.

8- Presence of enzymes inhibitor:
presence of enzyme inhibitor decreases
or stops the enzyme activity.
Enzyme inhibitors may be:
1- Competitive inhibitors.
2- Non competitive inhibitors.

Activity
• 1- High Km enzyme:
A) has high affinity to its substrate.
B) Need high conc. of its substrate to reach its Vmax .
C) has high max. velocity .
D) like hexokinase.
• 2- Optimum pH:
A) it is the same for all enzymes
B) it is acidic for pepsin enzyme.
C) at which the enzyme act at lowest rate
D) 41
the enzyme is stable under its marked changes
• 1- What is meant by Km?
• 2- What is significance of low Km
• 3- What is significance of high Km
42
Catalytic Protein
(Enzyme) Chemistry
ILO-3
Describe and explain enzyme Kinetics.

❑ Kinetics are concerned with the rates of
reactions.
❑ Living organism maintains its steady state by
adjusting reaction rates in response to the
environment and to hormonal controls.
❑ The study of the rate at which an enzyme acts
is called enzyme kinetics.
Michaelis-Menten hyperbolic plot
❑ Enzyme kinetics are studied by plotting the initial
velocity (Vi) on the Y axis and the substrate
concentration [S] on the X axis
❑Eventually, the enzyme becomes saturated with
the substrate and the reaction attains its maximal
velocity (Vmax).
❑ Any further increase in [S] does not affect the
velocity of the reaction.
Vmax, the maximum rate (plateau)
Vmax
Vmax is approached Vmax/2
asymptotically
Km
0 [S]
➢ The substrate concentration that produces
½ Vmax is the Michaelis-Menten constant,
Km.
➢ The produced curve attains the shape of a
rectangular hyperbola. So the curve is called
Michaelis- Menten hyperbolic curve.
➢ It is hard to draw accurately and hard to
determine Vmax and Km precisely.
A plot of Michaelis-Menten hyperbolic
plot is not accurate enough to derive good
Km & Vmax.
Computer analysis is done.
Reciprocal Plot
A double reciprocal plot or
Lineweaver-Burk plot is linear and more
eye-appealing for presentation.
mathematically = “linear transformation”
Lineweaver-Burk plot
Plotting the reciprocals of Vi and [S] (plotting 1/Vi on

the Y axis and 1/[S] on the X axis) yields a ‘double-
reciprocal” or Lineweaver-Burk plot.
➢ Vmax is determined by the point where the line crosses

the Y axis the [S] is infinite at that point.
➢ Km is easily determined from the intercept on the X axis.
➢ The curve is linear, so it is widely used as it is easy to
draw and provides a more precise way to determine Vmax
and Km.
1/v
intercept slope =
= 1/Vmax Km/Vmax
-1/Km
1/[S]
Catalytic Protein
(Enzyme) Chemistry
LO 4-
Correlation the knowledge to a clinical

situation
Case Report and Clinical
Correlates
• The parietal cells of the stomach secrete HCl into the lumen, resulting
in a pH 1 - 2. This strongly acidic environment is capable of
irreversibly denaturing most proteins. Many of the peptide bonds in
proteins would not be accessible to digestive proteases unless the
protein was denatured. Pepsin, a digestive protease, is an
exceptional enzyme because its pH optimum is approximately 1.6 and
it is active in the acidic environment of the stomach.
• As denatured dietary proteins pass into the intestinal lumen, the pH

of the gastric juice is raised above 6 by secretion of bicarbonate from
the exocrine pancreas. At this higher pH, chymotrypsin and other
proteases from the pancreas can act on the denatured proteins.
Summary and rap up
Discussion
10 minutes
Brain storming question
(Case Scenario)
In winter, Ahmed suffers frequently from common cold.

His body temperature raises. He feels fatigue. How
does fever affect his body metabolism?
Feed back
References
• Chatterjea’s Textbook of Medical Biochemistry,

8th edition.
• Vasudevan's Textbook of Biochemistry For

Medical Students, 6th Edition.
• BRS Biochemistry, Molecular Biology, and

Genetics, 5th Edition.

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Faculty of

Mechanism of Enzyme Action

➢ Mechanism of enzyme action.

➢ Factor affecting enzyme activities.

At the end of the lecture, the students should be able to:

• Explain the mechanism of enzyme action

• List, identify and describe different factors affecting

• Describe and explain enzyme Kinetics.

• Correlate their knowledge to a clinical situation.

• Go in a trip of a grilled meat in our

Explain the mechanism of enzyme action

2- The activated complex (ES) cleaved to the products (P)

3- In conjugated protein enzymes: coenzyme acts as an

1. Substrate approaches active site

- In this model, the active sites of the unbound enzyme is

In this model, the enzyme changes shape on

1- Enzymes increase the rate of reaction by

2- The activation energy is needed to overcome

The product no longer fits the binding site and is released.

List, identify and describe different

• Concentration of ion activators

The rate of enzyme action is directly proportional

-The rate of reaction increases as the substrate concentration

At Vmax, the enzyme is completely saturated with the substrate any

• E.g. Hexokinase acts on glucose at low concentration

• Enzymes with high Km: they have low affinity to the

• E.g. Glucokinase enzyme acts on glucose at high

- Rate of reaction increases gradually with

- The optimum temperature is 37-40 °C in

1- Increase of temperature increase the initial energy of

2- Increase of collision of molecules: more molecules

* After the optimum temperature, the rate of reaction

- Each enzyme has an optimum PH at

• E.g. Optimum PH of pepsin = 1.5 - 2

• Optimum PH of pancreatic lipase = 7.5 - 8

• Optimum PH of salivary amylase = 6.8

1- The enzyme activity depends on the ionization

2- Marked change in PH will cause denaturation

In the conjugated enzymes that need

Enzymes are activated by ions:

1- Chloride ion activate salivary amylase

2- Calcium ion activate thromobokinase

• In an enzymatic reaction, the rate of

• This is due to:

1- The decrease in substrate concentration.

2- The accumulation of the end products.

3- The change in PH than optimum PH.

Enzyme inhibitors may be:

2- Non competitive inhibitors.

A) has high affinity to its substrate.

B) Need high conc. of its substrate to reach its Vmax .

C) has high max. velocity .

A) it is the same for all enzymes

B) it is acidic for pepsin enzyme.

C) at which the enzyme act at lowest rate

• 2- What is significance of low Km

• 3- What is significance of high Km

Describe and explain enzyme Kinetics.

Vmax is approached Vmax/2