Enzymes

Compiled by :
dr. Santoso

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme activity
D.Regulation of enzyme activity

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme activity
D.Regulation of enzyme activity

What Are Enzymes?

Most enzymes are

Proteins (tertiary
and quaternary
structures)
Act as Catalyst to
accelerates a
reaction
Not permanently
changed in the
process

Enzymes

Are specific for

what they will
catalyze
Are Reusable
End in ase
-Sucrase
-Lactase
-Maltase

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme activity
D.Regulation of enzyme activity

How do enzymes Work?

Enzymes work by
weakening bonds
which lowers
activation energy

How do Enzymes Affect Reaction Rates?

Enzymes affect the rates
of reactions by lowering
the amount of energy of
activation required for
the reactions to begin.
Therefore processes can
occur in living systems at
lower temperatures or
energy levels than it
would require for these
same reactions to occur
without the enzymes
present.

How do Enzymes Bind to Substrates

There are two proposed methods by
which enzymes bind to their substrate
molecules:

Lock and Key Model

Induced-Fit Model

Enzyme-Substrate Complex

The substance
(reactant) an
enzyme acts on is
the substrate
Substrate

Joins

Enzyme

11

Active Site

A restricted region of an enzyme

molecule which binds to the substrate.
substrate
Active
Site
Substrate

Enzyme

12

Lock and Key Model

Active site
S2
enzyme
S2
S1
SUBSTRATE
MOLECULES

S1

enzyme

Products
P P
enzyme

ENZYME SUBSTRATE
COMPLEX

Enzyme returns from the reaction unchanged

and can now react with more substrate.

Induced Fit

A change in the
shape of an
enzymes active
site
Induced by the
substrate

14

Induced Fit
A change in the configuration of an
enzymes active site (H+ and ionic
bonds are involved).
Induced by the substrate.

substrate

Active Site
Enzyme

induced fit
15

Enzyme Cooperativity
Some enzymes have
multiple active site. It
has been observed that
when one substrate
molecule binds to a
single active site in the
inactive form or tense
state of the enzyme, a
configurational change
occurs in the other
active sites making them
more receptive to other
substrate molecules.

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Kinetic of enzyme activity
D.Factor affecting enzyme activity
E. Regulation of enzyme activity

Enzyme Kinetics
Expression for enzyme catalyzed reaction:

E+S

k1
k-1

ES

k2

E+P

Michaelis-Menten Equation
V0 = Vmax[S] / KM + [S]
Rate increase with [S]
Rate levels off as
approach Vmax
More S than active
sites in E
Adding S has no effect

 Vmax occurs when

enzyme active sites are
saturated with
substrate
Km (Michaelis-Menten
constant) reflects
affinity of enzyme for
its substrate
smaller the Km, the
greater the affinity an
enzyme has for its
substrate

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme activity
D.Regulation of enzyme activity

What Affects Enzyme Activity?

Three factors:
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors

22

1. Environmental Conditions
1. Extreme Temperature are the most
dangerous
high temps may denature (unfold)
the enzyme.
2. pH (most like 6 - 8 pH near neutral)
3. Ionic concentration (salt ions)

23

Temperature
All enzymes have an
optimum temperature at
which they work best.
If you observe the
enzymes activity below
the specific
temperature it will
steadily increase until it
reaches the optimum.
After the optimum
temperature is reached
the enzymes activity
drops dramatically due
to denaturing.

Depending on the species, the

range of optimum activity is very
broad. Above is a comparison of
human enzyme activity with that
of
bacteria found in hot springs and
oceanic vents.

pH
All enzymes have an
optimum pH at which
they work best. If
the pH falls below or
rises above the
optimum value,
enzymatic activity
decreases
as a result of
denaturing.

In the human bodys digestive tract

there are variations in pH from area
to area. The stomachs juices pH
is around 2 (acidic), the enzyme pepsin
found in the gastric juices has
optimum activity at a pH of 2. The small
intestines juices pH is around 8 (basic).
The enzyme trypsin found in the small
intestines juices has optimum activity at a pH
of 8.

Substrate Concentration
The concentration of substrate also has an affect on the rate
of enzyme activity. If the concentration of substrate is
increased while the concentration of enzyme is constant, the
level of enzyme activity will increase until a point of saturation
is reached. At this point there are no enzymes available to
react with excess substrate and the rate of the reaction
stabilizes. No matter if you continue to add substrate, the
reaction rate will not increase!
Rate of Reaction

Point of Saturation, all active

sites are filled with substrate.

Increasing Substrate Concentration

2. Cofactors and Coenzymes

Inorganic substances (zinc, iron) and
vitamins (respectively) are sometimes need
for proper enzymatic activity.
activity
Example:

Iron must be present in the quaternary

structure - hemoglobin in order for it to
up oxygen.

pick

27

Coenzymes are bound at the active site in order to

interact with the substrate and play an essential role in
the catalysed reaction.
They act as carriers of a variety of chemical groups.

Most water-soluble vitamins are components of coenzymes

Vitamin

Coenzyme

Deficiency

Thiamine (B1)

Thiamine
pyrophosphate

Beriberi (weight
loss,other problems

Riboflavin (B2)

FAD+

Mouth lesions, dermatitis

Nicotinic acid
(niacine)

NAD+

Pellagra (dermatitis,
depression)

Pantohtinic acid

Coenzyme A

Hypertension

Biotin

Biotin

Rash, muscle pain

3. Enzyme Inhibitors

Specific for an enzyme

Can be reversible or non-reversible
Competitive inhibitors
Non-competitive inhibitors

Competitive inhibitors
chemicals that resemble an enzymes
normal substrate and compete with
it for the active site.
site
Substrate
Competitive inhibitor

Enzyme

31

Noncompetitive Inhibitors
Inhibitors that do not enter the active site,
site but
bind to another part of the enzyme causing the
enzyme to change its shape,
shape which in turn
alters the active site.
site
Substrate
active site
altered

Enzyme

Noncompetitive
Inhibitor

32

Competitive vs. Non-competitive inhibitors

A.Introduction
B. Mechanisms of Enzymatic reaction
C. Factor affecting enzyme activity
D.Regulation of enzyme activity

Enzyme activity is regulated by four

different mechanisms*
(1)
(2)
(3)
(4)

Allosteric control
Covalent modification
Proteolytic activation
Stimulation and inhibition by control proteins

* changes in enzyme levels due to regulation of

protein synthesis or degradation are additional,
long-term ways to regulate enzyme activity

Allosteric regulation of enzyme activity

Allosteric regulation = the activation or
inhibition of an enzymes activity due to binding
of an effector molecule at a regulatory site
that is distinct from the active site of the
enzyme
Allosteric regulators generally act by increasing
or decreasing the enzymes affinity for the
substrate

Allosteric regulation

Many allosterically controlled enzymse show

quaternary structure

Covalent modification regulates the catalytic

activity of some enzymes
Modifying
group

Enzyme

Inactive Enzyme

Modifying
group

Enzyme

Active Enzyme

Can either activate it or inhibit it by altering the

conformation of the enzyme or by serving as a
functional group in the active site.

Phosphorylation - an example of regulation by

reversible covalent modification of the enzyme
OH ATP ADP
+
+

O
O P O
O-

Inserting a negatively charged phosphate group

into the appropriate location in an enzyme can
induce a conformational change in the enzyme that
either increases, or decreases, its activity.

Top 5 reasons why phosphorylation is used

to regulate enzyme activity:
1. Phosphorylation is rapidly reversible, making it possible to quickly
switch between active and inactive forms of an enzyme.
2. Phosphorylation is relatively inexpensive since it does not require
the synthesis of new protein molecules.
3. Results in large Grxn for the phosphorylation reaction.
Phosphorylation can shift the conformational equilibrium of a
protein by a factor of 104.
4. Phosphorylation/dephosphorylation is rapid and its timing can be
adjusted to meet the physiological needs of the cell.
5. Phosphorylation effects can be rapidly amplified via a kinase
cascade.

Summary: Covalent modification

1. Covalent modification allows an enzyme to be rapidly
activated or inactivated
2. With covalent modification, regulation of a enzyme
activity is achieved at low energy costs to the cell (i.e.
regulation does not require synthesis of a new enzyme or
inhibitory protein).
3. Phosphorylation is a good example of how enzymes are
activated and inactivated by covalent post-translational
modifications

Proteolytic activation

Proteolytic activation
.

(inactive)

Propeptide

P ro p e p tid e

Enzyme

Enzyme
(active)

Proteolytic
Enzyme

Such as those involved in protein digestion, blood clotting, and bone

and tissue remodeling, must be kept in a completely inactive state
until they are needed. These enzymes are synthesized as inactive
precursors (known as zymogens or proenzymes) and activated when
needed by proteolytic cleavage of a specific peptide bond in the
zymogen.

Regulation of digestive enzymes

Val

(Asp) Lys Ile Val

4
Trypsinogen
Enteropeptidase

Val

(Asp)4 Lys +

Ile Val
Trypsin

Proteolytic activation
of digestive enzymes

Digestion of proteins requires simultaneous

activation of several digestive enzymes.
This is achieved by synthesizing the digestive
enzymes as inactive zymogens that are activated
by specific proteolysis by trypsin.
Trypsin is activated by enteropeptidase
catalyzed proteolysis of a unique lysineisoleucine peptide bond (this is the master
switch that turns on the activation of the
digestive enzymes).

Pepsinogen is converted to pepsin by

autocatalytic proteolysis at pH 2

pepsinogen
(inactive)
Secretion into
stomach (pH 2)
autocatalytic
cleavage of
pepsinogen after
amino acid 44

pepsin
(active)

Pepsinogen has a low

amount of activity at
pH 2, allowing it to
cleave the peptide
bind between amino
acids 43 and 44 to
generate pepsin, that
is much more active
than pepsinogen.

Zymogen

Active Enzyme

Function

Pepsinogen
Chymotrypsinogen
Trypsinogen
Procarboxypeptidase
Proelastase
Prothrombin
Fibrinogen
Factor VII
Factor X
Proinsulin
Procollagen
Procollagenase

Pepsin
Chymotrypsin
Trypsin
Carboxypeptidase
Elastase
Thrombin
Fibrin
Factor VIIa
Factor Xa
Insulin
Collagen
Collagenase

protein digestion
protein digestion
protein digestion
protein digestion
protein digestion
blood clot formation
blood clot formation
blood clot formation
blood clot formation
plasma glucose
homeostasis
component of skin and
bone remodeling
processes during
metamorphosis, etc.

Digestive enzymes, blood clotting enzymes, and enzymes

involved in bone and tissue remodeling catalyze reactions
that would be disastrous if they occurred at
inappropriate times or locations.
For example, if proteolytic digestion of proteins
occurred in the pancreas, they would start digesting
the pancreas itself. Similarly, if blood clotting factors
are activated when they arent needed, they will
initiate blood clotting throughout the body.
So, they are synthesized as inactive zymogens and are
stored in this inactive state until they are needed.

Blood clot formation - an example of zymogen activations

Intrinsic Pathway
Damaged Surface
Kininogen
Kallikrein

XII

XIIa

Extrinsic Pathway
Trauma

XIa

XI

VII

VIIa
Tissue
IXa
VIIIa factor

IX

Xa
Va

Prothrombin

Thrombin

Fibrinogen

Fibrin
XIIIa
Cross-linked
fibrin clot

Blood clotting is an excellent example of a proteolytic

cascade designed to amplify an external signal (e.g.
trauma) and evoke a rapid response (blood clot
formation).
Thrombin itself is inhibited by antithrombin (a serpin).
This provides the body with a mechanism to prevent
random blood clot formation beyond the site of injury.

Proteolytic cleavage differs from phosphorylation

1. It can occur outside of cells, since ATP is not

needed to convert the zymogen into the active
form of the enzyme.
2. It is not a reversible reaction. Inactivation of the
active enzyme must occur by either degradation of
the enzyme or by inhibition (e.g. due to the binding
of an inhibitory protein to the active enzyme).

Stimulation and inhibition by control proteins

Inhibitory
Protein
Enzyme
(active)

Enzyme
(inactive)
Inhibitory
Protein

Some enzymes have regulatory proteins that bind to them

and regulate their activity. cAMP-dependent protein kinase
is one examples of this type of regulation.

Serpins - An example of inhibition by control proteins

Once trypsin is activated, we
need a mechanism to turn it
off when it is no longer
needed. Pancreatic trypsin
inhibitor is used to shut off
trypsin activity

Trypsin (orange) bound to bovine pancreatic trypsin inhibitor

(violet). His 57, Asp 102, Gly 193, and Ser 195 in the
active site of trypsin are shown in green, red, cyan, and blue,
respectively. Lys 15 in BPTI forms a salt bridge with Asp 189
in trypsin in the trypsin:BPTI complex. Binding of bovine
pancreatic trypsin inhibitor is essentially irreversible.

pancreatic trypsin inhibitor

binds very tightly to trypsin
pancreatic trypsin inhibitor
is a member of a class of
proteins known as serine
protease inhibitors (serpins).
Serpins are polypeptides
that inhibit serine proteases
by binding to the active
sites of these enzymes.

Elastase is inhibited by 1-antitrypsin

1-antitrypsin inhibits elastase, a
serine protease that is responsible
for remodeling collagen.
Individuals in which Glu 342 in 1antitrypsin is replaced by a lysine
secrete only 15% of the normal
levels for 1-antitrypsin, resulting
in uncontrolled elastase activity and
the breakdown of the alveolar walls
in the lung.

Note that although serpins are tight binding

inhibitors or serine proteases, they do not form
covalent bonds with the serine proteases.
In other words, binding of serpins to serine
proteases does not involve the formation of covalent
bonds between the serpins and the serine proteases.

Summary of regulatory mechanisms

1. Allosteric regulation
ATP activation/CTP inhibition of ATCase sigmoidal kinetics
cAMP activation of cAMP-dependent protein kinase

2. Reversible covalent modification

Phosphorylation
Ser/Thr protein kinases, Tyr kinases, kinase cascades

3. Proteolytic activation
Digestive enzyme, blood clotting factors

4. Protein activators and inhibitors

Serpins

Regulating the rates of enzyme-driven

reactions
Cells use inhibitors and activators to turn off and
on enzymes
Many enzymes are controlled by an allosteric site
remote from the active site

Feedback inhibition
Many enzymes are actually regulated by the
end products of the reaction they catalyze

Start of
pathway

Enzyme 1

Intermediate

Enzyme 2

Intermediate

Enzyme 3

Presence of product inhibits enzyme 1

This prevents too much product from being made

Product

An example of Feedback inhibition

This example demonstrates how an
end product can inhibit the first
step in its production. Isoleucine
binds to the allosteric site of
threonine deaminase and prevents
threonine from binding to the active
site because the shape of the active
site is altered. When the level of
isoleucine drops in the cells
cytoplasm, the isoleucine is removed
from the allosteric site on the
enzyme, the active site resumes the
activated shape and the pathway is
cut back on and isoleucine begins
to be produced.

Klasifikasi Enzim
Sistem Penamaan Enzim
Spesifitas Enzim
Macam-macam Bentuk Enzim

Klasifikasi Enzim
5 kelas
Oksidoreductase
Transferase
Hidrolase
Isomerase
Ligase

Sistem Penamaan Enzim

Reaksi dan enzim yang mengkatalisisnya membentuk enam
kelas (4 hingga 13 subkelas)
2 bagian :
Nama pertama substratnya
Nama kedua tipe reaksi yang dikatalisis.

Informasi tambahan, dituliskan dalam tanda kurung di bagian

akhir;
misalnya enzim yang mengkatalisis reaksi L-malat + NAD + = piruvat
+ CO2 + NADH + H+ diberi nama 1.1.1.37 L-malat:NAD+
oksidoreduktase (dekarboksilasi)

Setiap enzim mempunyai nomor kode (EC) yang menandai tipe

reaksi berkenaan dengan
kelas (digit pertama)
subkelas (digit kedua)
subsubkelas (digit ketiga)

Spesifitas Enzim
Enzim biasanya sangat spesifik dalam aksinya.
Beberapa kespesifikan yang dimiliki oleh enzim antara lain :

Kespesifikan
Kespesifikan
Kespesifikan
Kespesifikan

geometrik
reaksi
optik.
organella

Macam-macam Bentuk Enzim

Proenzim
Bentuk enzim yang inkatif
Isozim
Bentuk enzim berbeda yang mengkatalisis reaksi kimia yang
sama. Isozim ini berasal dari duplikasi gen
Alosterik enzim
Bentuk enzim yang diatur dengan mekanisme alosterisme.
Ada 2 sisi :
Sisi aktif
Sisi regulatorik

Enzim plasma

Enzim plasma non

fungsional
fungsional
Konsentrasi Lebih
tinggi
dalam Secara normal, konsentrasi
dalam

plasma

dibandingkan di

plasma

dengan dijaringan

dalam

plasma

sangat

rendah dibandingkan dengan

di dalam jaringan

Fungsi

Jelas

Tidak jelas

Substrat

Berada dalam darah

Tidak ada dalam darah

Tempat

Hepar

Diberbagai

macam

organ

sintesis

seperti hepar, jantung, otak

Contoh

Factor pembekuan

dan otot rangka

ALT, AST, CK, LDH, alkaline

Lipoprotein lipase

phospatase, amilase

Pseudocholine
esterase

THANK YOU