Enzymes Lecture 2

Objectives
–Definition of the Reaction velocity (reaction rate or
speed)

–Factors affecting reaction velocity

–Enzyme Kinetics

–Michaelis-Menten Equation

–Lineweaver-Burk plot

– Enzyme inhibitors

–Enzyme inhibitors as drugs

 Reaction velocity (V)

• The velocity of a reaction (V) = the rate of

the reaction
• It is the number of substrate
molecules converted to product per
unit time.
• It is usually expressed as μmol of the
formed product per minute (μmol/min)
• Factors affecting reaction velocity

1) Substrate concentration (and nature)

2) Enzyme concentration

3) Temperature
4) pH
 1. Effect of temperature on
reaction velocity
• Increasing the temperature increases the
reaction velocity, until a peak velocity
is reached at the optimum temperature
–Explanation:
–Increasing the temperature increases the
number of molecules having enough
energy to pass over the energy barrier of
the transition state & form the product
• Increasing the temperature above the
optimum temperature decreases the
reaction velocity due to denaturation of
the enzyme
2. Effect of pH on reaction
velocity
• Each enzyme has an optimum pH at which the
enzyme function in the body
• At optimum pH, enzyme activity is maximal
• Optimum pH varies for different enzymes
e.g.,
– Pepsin optimum pH is 2
– Alkaline phosphatase optimum pH = 9
• High changes in pH will result in enzyme
denaturation
 How can pH affects the
reaction velocity?
• pH affects the reaction velocity:
1. Small change in pH, change the ionization of the active site
of the enzyme (&/or the substrate): Catalytic activity
requires that enzyme & substrate have specific chemical
groups in either ionized or deionized states in order to interact
2. High changes in pH (extremes of pH), causes denaturation
of the enzyme
3. Effect of enzyme concentration
on reaction velocity

• Reaction velocity is directly

proportional to enzyme
concentration
• Therefore, an increase in
enzyme concentration increases
reaction velocity.
4. Effect of substrate concentration
on reaction velocity
•Increasing the substrate
concentration increases the
reaction velocity, until a
maximal velocity (Vmax)
is reached
•So, When the Vmax can
be reached?
When the enzyme active site is
fully saturated with the substrate.
•So, after this any increase in
substrate concentration will not
affect reaction velocity
Equal amount of
Enzymes
But different
amount of
substrate

Vmax is the maximal

reaction rate or velocity of
an enzymatically catalyzed
reaction when the enzyme
is saturated with its
substrate
Enzyme
Kinetics
 Michaelis-menten equation

• Two scientists designed an

equation to describe enzyme
kinetics
• Based on steady state theory
• From this you can predict
the enzyme kinetics
 Intro to Enzyme
Kinetics
kf
E+S ES kcat E+P

kr

Variables Parameters
[E]: free enzyme molecules kf , kr , kcat : reaction rates
[S]: free substrate molecules
[ES]: enzyme-substrate complexes
[P]: free product molecules
 Michaelis-Menten Equation

• Michaelis and Menten designed an equation to describe

enzyme kinetics and it is called Michaelis-Menten Equation

• Michaelis-Menten Equation explains how the velocity varies

with substrate concentration:

Vmax [S]
vo =
K
m
+
V0 = initial reaction velocity Vmax = maximal velocity
Km = Michaelis constant = (k-1+k2)/k1 [S] = substrate concentration

[
That’s why we want to measure the
Vo (initial velocity of the enzyme)
Km is numerically equal to substrate
concentration when the reaction velocity
equals ½Vmax..
Km reflects the enzyme affinity for its
substrate
K
m

Which enzyme is
more efficient and has
a higher affinity ?
 Let’s revise the equation

Vmax [S]
vo =
K
m
• V0= initial reaction velocity
+
– Is the reaction velocity measured at the start of a
reaction when very little product (P) has been made
[
• Vmax = maximal velocity S
]
– Is the maximal rate of the reaction
– Is reached by increasing the concentration of the
substrate
– At Vmax, the enzyme is saturated with the substrate
• Km = Michaelis constant
– Different enzymes have different Kms
– Km is numerically equal to substrate concentration when the
reaction velocity equals ½ Vmax
– Km reflects the enzyme affinity for its substrate:
• Small Km means high affinity of the enzyme for the substrate
(because low substrate concentration is needed to ½ saturate the
enzyme, thus reaching a velocity = ½ Vmax)
• Large km means low affinity of the enzyme for the
substrate (because high substrate concentration is needed to ½ saturate
the enzyme, thus reaching a velocity = ½ Vmax)
For most enzymes (which follow the Michaelis-Menten kinetics)
the shape of the curve plotted between the initial reaction
velocity (V0) & the substrate concentration [S] is hyperbolic
Does this Rule Apply to
all enzymes ??
Allosteric Enzymes has Sigmoidal curve
Michaelis-Menten Equation
Has a problem

The Plateau
phase
Cannot accurately
determine Vmax
What can we do to fix The
problem ?
Lineweaver – Burk Double Reciprocal
Plots
The Michaelis-Menten equation can be recast into a linear
form
 Lineweaver-Burk plot
• Is a straight line obtained when 1/V0
is plotted versus1/[S]. (Double
reciprocal)
• The intercept on the x axis is
equal to -1/km & the intercept on
the y axis is equal to 1/Vmax
• Lineweaver-Burk plot can be used
to:
• calculate Km & Vmax
• Determine the mechanism of action
of enzyme inhibitors
 Inhibition of enzyme activity
Enzyme Any substance that decrease the velocity of
Inhibitor enzyme-catalysed reaction
Types

Reversible Irreversible
Inhibitors Inhibitors
-Weakly bind enzyme -Strongly bind enzyme
-Enzyme regain activity -Enzyme cannot
regain activity
Competitive Non-Competitive e.g Heavy metal ions
 Competitive inhibition (substate
concentration effect)
S S
I
S
I
S
S S
I
S
I
S
S
I
I

But as when substate is high in

Substrate is low in concentration , concentration , enzymes will bind to S
competitive inhibition , so Velocity will and Vmax wont change
be lower
 Non- Competitive inhibition (substate
concentration effect)
S
S
S
S
I

S
S
I
I

But since the inhibitor is working and

Is the affinity (binding ability of the effecting the reaction will we have the
enzyme effected ) i.e is Km going to be same product per time produced i.e
effected ? Vmax will be effected?
 Competitive Noncompetitive
•Vmax : not changed •Vmax : decreased
•K m : increased •K m : not changed
 Enzyme inhibitors as
drugs
• Antibiotics as Penicillin & amoxicillin inhibit
enzymes responsible for cell wall synthesis
in bacteria.
• Aspirin is an anti-inflammatory agent because it
inhibits an enzyme called cyclooxygenase
• Statins competitively inhibit HMG-CoA reductase
which synthesizes cholesterol, so statins are
antihyperlipidemic drugs (cholesterol lowering)
 Recommended Readings

• Lippincott’s illustrated reviews biochemistry

• https://www.khanacademy.org/science/biol
ogy/ener
gy-and-enzymes/enzyme-regulation/a/basics-of-
enzyme-kinetics-graphs
• https://www.youtube.com/watch?v=y43pIHUtjeM