Anda di halaman 1dari 45

Factors that

affect Enzyme
Activity
Enzyme activity

• Is a measure of the rate at which an


enzyme converts substrate to products in
a biochemical reaction.
• Four factors affect the enzyme activity:
– Temperature
– pH
– Substrate concentration
– Enzyme concentration
2
jdejuan
Temperature
• Is the measure of kinetic energy of
molecules
•  temperature
• Higher temperatures
mean molecules are of an
moving faster and enzymatically
colliding more catalyzed
frequently. reaction =  rate
• This concept applies (velocity) of the
to collision between
reaction.
substrate molecules
and enzymes.
3
jdejuan
Temperature Optimum
temperature
• When the temperature
Increased number
increases beyond a
of enzyme-
certainsubstrate
point, the energy
collision
begins to cause
disruption in the tertiary
structure of the enzyme;
denaturation is occurring.
• Optimum temperature is
Denaturation due
the temperature at
to excess heat
which an enzyme exhibits
maximum activity. 4
jdejuan
Temperature
• For human
example, enzymes,
a certainthe optimum
human temperature
enzyme’s
isoptimum
around 37°C.
Optimum
A personis with
body
temperature 37°C,fever,
if the40°C, can

temperature temperature
number of still increases, there will beitenzyme
be a life threatening situation because can
enzyme- substrate
denaturation.
initiate enzyme denaturation.
collision Denaturation due
to excess heat
= loss of function of critical
enzymes, particularly those of the
central nervous system, which can
result in dysfuction sufficient
enough
35 °C to 37
36 °C cause
°C DEATH.
38 °C 39°C 40°C
5
jdejuan
pH
•• pH of an enzyme’s
Optimum environment
pH is the pH at which thecan
enzyme
affect
exhibitsitsmaximum
activity. activity. Each enzyme
has acharge
• The characteristic optimum
on acidic pH, amino
and basic which
usually falls within the range of 7.0 – 7.5,
acids located at the active site depends
with some exceptions like the digestive
on pH.
enzymes – Pepsin in the stomach is active at
• Small changes
pH – 2.0 in pHin(less
and Trypsin thethan
small1intestine
unit) is
can result
active at pHin 8.0.
enzyme denaturation and
• subsequent
Biochemical loss of catalytic
buffers activity.
help maintain the
optimum pH for an enzyme.
6
jdejuan
pH
A graph showing the effect of pH on the
rate of enzymatic activity with pH 7 as
the optimum pH.
Maximum rate

Optimum
pH
5 7 9
7
jdejuan
Substrate Concentration
This graph shows the change in enzyme activity
•with:
When the concentration of an enzyme is kept
 constant
Substrateand concentration
concentration of substrate
+ constant is
temperature,
increased, the enzyme
pH, and enzyme activity pattern increases
concentration
=up
enzyme activity substrate
to a certain remains constant when aremains
concentration certain
substrate concentration is reached.
constant.
Maximum reaction rate

Rate approaches
maximum
Saturation
Curve
8
jdejuan
Substrate Concentration
• What limits enzymaticActive
activitysite
to a certain
Substrate
maximum
Next value?
– As substrate concentration increases, toProducts
Substrate a point
Enzyme
where it eventually reached where the enzyme
capabilities are used to their maximum extent.
– The rate remains constant from this point on.
Each substrate must occupy an enzyme active
site in a definite amount of time, and the
products must leave the site before the cycle
can be repeated – then the incoming
substrate molecules must “wait for its turn”
for an empty active site.
9
jdejuan
Substrate Concentration
• The rate which an enzyme accepts
substrate molecules and releases product
molecules at substrate saturation is given
by its turnover number.
• An enzymes turnover number is the
number of substrate molecules
transformed per minute by one molecule of
enzyme under optimum conditions of
temperature, pH, and saturation.
10
jdejuan
Substrate Concentration
Enzyme Turnover Number Reaction Catalyzed
(per minute)

Carbonic anhydrase 36, 000, 000 CO2 +H2O H2CO3

catalase 5, 600, 000 2H2O2 2H2O + O2

cholinesterase 1, 500, 000 Hydrolysis of acetylcholine

penicillinase 120, 000 Hydrolysis of penicilin

Conversion of pyruvate to
Lactate dehydrogenase 60, 000 lactate
Addition of nucleotides to
DNA polymerase 1 900 DNA chains
11
jdejuan
Enzyme Concentration
• Reason
Because enzymes
: The are paying
cells avoid not consumed in the
the energy costs
ofreactions theyand
synthesizing catalyze, the cells
maintaining usually
a large work
force
keeps of the
enzyme molecules.
number of enzymes low
compared with the number of substrate
In general: In a reaction, the concentration of
molecules.
substrate is much higher than the enzyme.

SUBSTRATES
ENZYMES 12
jdejuan
Enzyme Concentration
• If the amount of substrate is kept constant
and the enzyme concentration is increased,
the reaction rate increases.
• Reason: more substrate molecules are
accommodated in a given amount of time.

PRODUCTS 13
jdejuan
Enzyme Concentration
A graph showing the change in reaction rate with
 Enzyme concentration + CONSTANT
a CHANGE IN ENZYME CONCENTRATION, while
Temperature, pH, and substrate
the TEMPERATURE, pH, and SUBSTRATE
concentration
CONCENTRATION  reaction rate
are =CONSTANT.

14
jdejuan
Example:
DescribeDetermine
the effecthow Enzyme
of each Activity
of the is
following
affected
changesbywould
Various Changes
have on the rate of reaction
that involves the SUBSTRATE UREA and the
ENZYME UREASE.
a. Increasing the Urea concentration
b. Increasing the Urease concentration
c. Increasing the temperature from its
optimum value to a value 10˚ higher than
this value.
d. Lowering the pH form the optimum value
of 5.0 to a value of 3.0 15
jdejuan
substrate Urea liver enzyme Urease

A. Increasing the Urea Concentration

Solution:
The enzyme activity will increase until
all of the enzyme molecules are engaged
with urea substrate.

16
jdejuan
substrate Urea liver enzyme Urease

b. Increasing the Urease Concentration

Solution:
The enzyme activity rate will increase
until all urea molecules are engaged with
urease enzyme.

17
jdejuan
c. Increasing the temperature from its optimum value
to a value 10˚ higher than this value.
Maximum
Reaction Rate

Solution:
At temperature higher than the optimum
temperature, enzyme activity will
decrease from that of the optimum
temperature.

18
jdejuan
d. Lowering the pH form the optimum value
of 5.0 to a value of 3.0
Maximum rate

Solution:
At pH values lower than the optimum pH
value, the enzyme will decrease from the
optimum pH.

3 4 5
19
jdejuan
ENZYME INHIBITION
• The rate of enzyme catalyzed reactions
can be decreased by a group of
• 3 modes of inhibition takes place:
substance called inhibitors.
• –An
Reversible
ENZYME competitive
INHIBITOR inhibition
is a substance
–that
Reversible noncompetitive
slows or inhibition
stops the normal catalytic
–function
Irreversible
of inhibition
the enzyme by binding to it.

20
jdejuan
Reversible Competitive Inhibition
• Enzymes are specific about the molecules
(substrates) they accept at their active site.
Molecular shape and charge distribution are
key determining factors in whether an
enzyme accepts a molecule.

Enzyme
 Substrates
21
jdejuan
Reversible Competitive Inhibition
•• When
A molecule
a competitive
that sufficiently
inhibitorresembles
binds to an
enzyme
enzyme’sactive
substrate
site the
in shape
inhibitor
andremains
charge
unchanged
distribution (nothatreaction
it can compete
occurs), but
withitsthe
physical
original presence
substrate at forthe
occupancy
site prevents
of the
a active
normal
site. substrate molecule from occupying
the site. Enzyme Already
– Inhibitor
Normal Enzyme – Reaction
Substrate Reaction
Substrates occupied!
No reaction =
no products
Enzyme Enzyme
Products Competitive inhibitor 22
jdejuan
Reversible Competitive Inhibition
• The formation of an enzyme-competitive
inhibitor complex is a REVERSIBLE process
because it is maintained by weak interactions
and with time (fractions of seconds) the complex
breaks up.
• The empty site is then available for a new
occupant. Another substrate and another
inhibitor will again compete for the empty
active site.
• Therefore, the active site of the enzyme binds
either on inhibitor or normal substrate on
random basis.
23
jdejuan
Reversible Competitive Inhibition
• If the inhibitor concentration is greater
than substrate concentration, the
inhibitor dominates the occupancy
process, resulting to decreases enzyme
activity.

INHIBITOR > SUBSTRATE =


 ENZYME ACTIVITY

24
jdejuan
Reversible Noncompetitive Inhibition
• Is a molecule that decreases Substrate
enzyme activity by binding to a
site on an enzyme other than the
active site.
• The substrate can still occupy the
active site, but the presence of the Enzyme
inhibitor causes a change in the
structure of the enzyme sufficient = No activity,
to prevent the catalytic groups at no reaction
Inhibitor
the active site form properly and no product
effecting their catalytic action.
25
jdejuan
Reversible Noncompetitive Inhibition
• Unlike the situation in competitive
inhibition, increasing the concentration
of substrate does not completely
overcome the inhibitory effect in this
case.
• However, lowering the concentration of
a noncompetitive inhibitor sufficiently
does free up many enzymes, which then
the enzyme return to normal activity.
26
jdejuan
Irreversible Inhibition
• Is a molecule that inactivates enzymes by
forming a strong covalent bond to an
amino acid side-chain group at the enzyme’s
active site.
• Inhibitors do not have structures similar to
that of the enzyme’s normal substrate.
• The inhibitor- active site bond is sufficiently
strong that addition of excess substrate does
not reverse the inhibition process, thus, the
enzyme is permanently deactivated.

27
jdejuan
Irreversible Inhibition
The substrate Irreversible
can’t enter inhibitor
Active Site
Substrate
Strong covalent
bond

Enzyme

28
jdejuan
Comparison between the 3 Enzyme
Inhibitors
ENZYME INHIBITORS

Reversible Reversible
Irreversible Enzyme
Competitive Enzyme Noncompetitive Enzyme
Inhibitor
Inhibitor Inhibitor
• A molecule closely • A molecule that binds to •A molecule, not the
resembling the a site on an enzyme that same shape like the
substrate, binds to is not the active site. substrates, that forms
active site and • The substrate still a covalent bond to a
occupies the active site part of the active site,
temporarily
but the enzyme can’t permanently
prevents substrates
catalyze due to the preventing substrates
from occupying the presence of the inhibitor. from occupying it.
enzyme.
29
jdejuan
Comparison between the 3 Enzyme
Inhibitors
ENZYME INHIBITORS

Reversible
Competitive Enzyme Irreversible Enzyme
Noncompetitive Enzyme
Inhibitor Inhibitor
Inhibitor
Substrate Covalent Inhibitor
can’t Substrate
bond
enter

New substrate Enzyme


and inhibitor will Enzyme
Enzyme
compete for the Inhibitor
active site
Inhibitor Substrate 30
jdejuan
REGULATION OF ENZYME
ACTIVITY
• Regulation of enzymes in a cell is a
necessity for many reasons.
• Regulation of enzyme activity is seen
in 2 certain situations- both are
involved in the concept of energy
conservation.
31
jdejuan
1. A cell that continually produces large
amounts of an enzyme for which substrate
concentration is always very low is wasting
energy.

The production
of enzyme
needs to be
“TURNED OFF”
Cell
32
jdejuan
2. A product of an enzyme-catalyzed reaction
that is present in plentiful (more than needed)
amounts in a cell, is a waste of energy if the
enzyme continues to catalyze the reaction that
produces the product.
Example: Cell A and B
needs 2 product AThe
and B enzyme
needs
of the enzyme- catalyzed
reaction.
to be
“TURNED OFF”.

Cell A Product A Product B Cell B 33


jdejuan
REGULATION OF ENZYME
ACTIVITY
• Many mechanisms exist by which
enzymes within a cell can be “turned on”
and “turned off”.
• In general terms, there is 3 such
mechanism:
– Feedback control associated with allosteric
enzyme
– Proteolytic enzyme and zymogens
– Covalent modifications 34
jdejuan
Allosteric Enzymes
• The term allosteric enzyme is an enzyme with 2 or
more protein chains (quartenary structure) and has 2
kinds of binding site (substrate and regulator)
• The term allosteric comes from the Greek allo, which
means “other” and stereos, which means “site or
space”.
• Characteristics:
1. All allosteric enzymes have quarternary
structure; that is, they are composed of 2 or
more protein chains.
35
jdejuan
2. All allosteric enzymes have two kinds of binding
sites: those for substrates, and those of regulators.
Active site / binding
site for substrates
Substrate

Allosteric
enzyme Allosteric
Binding site for Regulator
Regulator
3. Active and regulatory binding sites are distinct
from each other in both location and shape. Often,
the regulatory site is on one protein chain and the
active site is another. 36
jdejuan
4. Binding of a molecule at the regulatory site
causes changes in the overall three-dimensional
structure of the enzyme, including the structural
changes at the active site.
Active site / binding
site for substrates

Allosteric
enzyme Allosteric
Regulator
Binding site for
Regulator
37
jdejuan
Allosteric Enzymes
• Substances that bind at regulatory sites of
allosteric enzymes are called regulators.
• There is positive and negative regulator.
• The binding of a positive regulator increases
enzyme activity, the active site is changed
such that it can more readily accept substrate
• Negative regulator (a noncompetitive
inhibitor) decreases enzyme activity, changes
the active site as such substrate is less readily
accepted.

38
jdejuan
Allosteric Enzymes
Positive Allosteric Negative Allosteric
Control Control
Substrate can
Unavailable Substrate
enter
active site
Negative allosteric
regulator
Substrate
Active siteenter
cannot
Allosteric site Inhibits/
Increases Substrate decreases
enzyme enzyme
activity
Positive Allosteric activity
Allosteric site
Regulator 39
jdejuan
Feedback Control
• Is a process in which activation or inhibition of
the first reaction in a reaction sequence is
controlled by a product of the reaction
sequence.
• Most biochemical process within cells take
place in several steps, rather than a single
step. A different enzyme is required for each
step sequence.

40
jdejuan
Feedback Control
Enzyme 2 Enzyme 3
A B C D
Enzyme 1

Biochemical process within a cell occurs in several


steps, each step is catalyzed by a different
enzyme, and the product of each step is the
substrate for the next enzyme.
Substrate A Substrate or Substrate or
products B product C
products products
Products D

Enzyme 1 Enzyme 2 Enzyme 3 41


jdejuan
Feedback Control
• What will happen in this reaction series if the
final product is a negative regulator (inhibitor)
of the first enzyme (enzyme 1)?
Substrate A Substrate / Substrate /
product B product C
products products
Product D

Enzyme Regulator /
1 Enzyme 3 Inhibitor of
Enzyme 2 Enzyme 1
Binding site No reaction, no products=
for regulator the reaction sequence is
not continued, the activity
stops. 42
jdejuan
Feedback Control
Feedback control
Inhibition of enzyme 1 by product D

Enzyme 1 Enzyme 2 Enzyme 3

A B C D

43
jdejuan
Feedback Control

• At low concentrations of D, the reaction


sequence proceeds rapidly.
• At higher concentrations of D, the activity
of enzyme 1 in inhibited (by feedback), and
eventually the activity stops.

44
jdejuan
Thank you for
listening!
Presented by:
Jessa O. De Juan
45
jdejuan