ENZYME-

BIOLOGICAL
CATALYST
Key words for today
• Enzyme – definition
• Enzyme catalysis – features
• Active site, substrate, ES complex
P Catalysis
r
o
t Structure
e
i
n Defence
F
u Transport and storage
n
c
t Regulation
i
o
n Movement
s
 H2 O2 H2 O + O2

a) No catalyst, b) with added Fe3+ salt, c) with

Enzyme As Catalyst
• All enzymes are proteins - with the exception of
some RNAs that catalyze their own splicing all
enzymes are proteins

• In general, names end with suffix “ase”

- tyrosinase (tyrosine), celullase (cellulose),
protease (protein), lipase (lipid)

• Enzyme: a biological catalyst

• enzymes can increase the rate of a reaction by a
factor of up to 1020 over an uncatalyzed reaction
• Catalysis:
- the process of increasing the rate of
chemical reactions

• Catalyst:
- the substance that facilitate in catalysis
 Enzyme As Catalyst
• Enzymes are catalysts:
1.increase the rate of a reaction
2.not consumed by the reaction
3.act repeatedly to increase the rate of reaction
4.enzymes often “specific” – promote only 1
particular reaction, others catalyze a family of
similar reactions
cellulase – cellulose as substrate
hexokinase – any 6 ring monosaccharide
-fructose, glucose
General Properties of Enzyme
• Higher reaction rates:
- the rates of enzymatically catalyzed reactions are
106 to 1020 > than uncatalyzed reaction
• Milder reaction rates:
- enzyme catalyzed reactions occur under relatively
milder conditions: < 100oC, atmospheric pressure and
nearly neutral pH- contrast with chemical catalysis
requires high temperature, pressures and extremes
pH.
• Greater reaction specificity:
- enzyme have greater degree of specificity to their
substrates and their products – rarely have side
products
Enzyme Catalysis
GENERAL FORMULA

E = enzyme
S = substrate
P = product
Enzyme Catalysis
Active
site/catalytic site -
part of enzyme to
which the
substrate binds
and the reaction
takes place
Substrate – a
reactant in an
enzyme-catalyzed
reaction
Product

Enzyme-substrate (ES) complex – the intermediate

formed when the substrate is bind at the active
site of an enzyme
Activity:

By using any resources available:

1.Discuss the application of enzyme in industries.
2.Discuss biological importance of enzymes.
ENZYME-
BIOLOGICAL
CATALYST
(2)
Key words for today
• Transition state theory
• Enzyme activity, rate of reaction
• Lock and key and induce fit binding models
• Cofactor, coenzymes, holoenzymes,
apoenzymes
Enzyme catalysis reaction
• Physically interact with their substrates to
effect catalysis
• E + S ES ES* EP E + P
• Where:
- E = enzyme
- ES = enzyme/substrate complex
- ES* = enzyme/transition state complex
- EP = enzyme/product complex
- P = product
Enzyme catalysis reaction
• E + S ES ES* EP E + P

• 1st step: enzyme binds to substrate molecule

to form an enzyme – substrate complex
• E+S ES

Enzyme
Enzyme catalysis reaction
• E + S ES ES* EP E + P

• 2nd step: Formation of the transition state

complex where the bound substance is
neither product nor reactant
• ES ES*, ES≠ES*
Enzyme catalysis reaction
• E + S ES ES* EP E + P

• 3rd step: Formation of the enzyme – product

complex ES* EP
Enzyme catalysis reaction
• E + S ES ES* EP E + P

• 4th step: Release of product EP E + P

Enzyme catalysis reaction
• Enzyme can only
work on one
substrate molecule at
a time
• No modification
during the reaction
• Once product is
release, enzyme is
available to accept
another substrate
molecule
Enzyme Catalysis
• Rate of reaction = reaction velocity (V)
- the rate of enzyme reaction is measured by
the rate of the appearance of products or the
rate of disappearance of substrates.
- d[P]/dT or d[S]/dT
mol product/min or mol substrate/min

• Enzyme activity
 1 unit (U) is the amount of enzyme that catalyses the
reaction of 1 mol of substrate per minute under
specified conditions.  Enzyme activity / rate of
reaction
Enzyme Catalysis
• The rate of a reaction depends on
its activation energy, G°‡
• an enzyme provides an alternative
pathway with a lower activation energy

• Activation energy – the energy

required to start a reaction/ the
amount of energy needed to get the
reactants to transition state

• Transition state – the intermediate

stage in a reaction in which the old
bonds break and new bonds are
formed
How enzyme work?
ES complex

• Transition state theory:

– The enzyme (E) must approach
the substrate (S), the substrate
attach to the active site through
noncovalent bond
– Formed the high energy (unstable)
ES complex
– In ES complex, the covalent bond
in substrate is in the process of
breaking while the EP complex is
forming.
Enzyme Catalysis - Example

• Consider the reaction

catalas
H2 O2 e H2 O + O2

a) No catalyst,
b) with added Fe3+
salt,
c) with added
catalase
 • (a)– a/e for the
reaction in the
absence of a catalyst

• (b) – a/e lowered in the

presence of an iron
catalyst

• (c) – energy diagram

for the catalase-
catalyzed breakdown
of H2O2

• (d) – energy diagram

for the noncatalysed
breakdown of H2O2 at
a/e – activation energy elevated temperature
Enzyme/substrate
Binding Modelsinteraction
• Two models have been developed to describe
formation of the enzyme-substrate complex

1. Lock-and-key model: substrate binds to that

portion of the enzyme with a complementary
shape

2. Induced fit model: binding of the substrate

induces a change in the conformation of the
enzyme that results in a complementary fit
Continue……

1. Lock-and-key model: substrate binds to that portion of the enzyme with a

complementary shape

2. Induced fit model: binding of the substrate induces a change in the conformation
of the enzyme that results in a complementary fit
Enzyme/substrate interaction
1. Lock and key model
- substrate (key) fits into a perfectly shaped space
in the enzyme (lock)
- lots of similarities between the shape of the
enzyme and the shape
of the substrate
- highly stereospecific
- implies a very RIGID
inflexible active site
 site is preformed and
RIGID
Enzyme/substrate interaction
2. Induced fit model (hand in glove analogy)
 count the flexibility of proteins
 substrate fits into a general shape in the enzyme,
causing the enzyme to change shape
(conformation);
close but not perfect fit
of E + S
 change in protein
configuration leads to
a near perfect fit of
substrate with enzyme
Active site
• Has specificity – can discriminate among possible
substrate molecules
- others recognize a functional group (group
specificity)
- only recognize one type of molecule (eg. D vs L
isomer)
(absolute specificity)
• Relatively small 3D region within the enzyme
- small cleft or crevice on a large protein
• Substrates bind in active site by weak non-covalent
interactions (hydrogen bond, hydrophobic and ionic
interaction)
 Active site
• The interactions hold the substrate in the proper
orientation for most effective catalysis

• The energy derived from these interactions –

binding energy

• Binding energy is used, in large part to lower the

activation energy and stabilize the transition state
complex (ES*)

• Each non-covalent interaction provides energy to

stabilize the transition state
Enzymes & cofactor
• Holoenzyme – an
enzyme in its
complete form
including
polypeptide(s) and
cofactor
• Apoenzyme –
enzyme in its
polypeptide form
without any necessary
prosthetic groups or
cofactors
Apoenzyme (inactive) + cofactor 
holoenzyme (active)
Enzymes and cofactor

• Enzymes require
chemical entity in order
to function properly
(assists an enzyme in
catalytic action)
• Cofactor – nonprotein
molecule that assist in
an enzyme catalytic
reaction
Enzymes & cofactor

Devided into 2 catogories: metal ions and coenzymes.

• Coenzyme
• Smaller organic or organometallic molecule derived from
vitamin
•  Cosubstrates = weakly bound to enzyme, temporarily
associated with enzymes, eg. NAD+ , FAD+
•  Prosthetic group – coenzymes that are covalently bound,
tightly bound to enzyme and always present, eg. heme in
catalase
Enzymes & cofactor

• Cofactor can be divided into 2 metal ions and

coenzymes. Some source limit the terms cofactor to
inorganic substance, eg. metal ion.
• People always say that loosely bound cofactor as
coenzyme but some call it as cosubstrate
ENZYME-
BIOLOGICAL
CATALYST
(3)
http://www.sumanasinc.com/webcontent/ani
mations/content/enzymes/enzymes.html
Key words for today
• You need to understand:
1. 6 classes of enzymes
2. Factors effecting enzyme reaction rate
3. Allosteric enzyme, effectors (positive and
negative), heterotropic and homotropic
allosterism
4. Isoenzyme and multienzyme
 Classification of Enzymes
• Have 6 categories

• Each enzyme has an official international

name ending with –ase and a classification
number

• Number consists in 4 digits (referred to a

class and subclass of reaction
Classification of Enzymes
Classification of Enzymes

Table 5.1, pg 136

Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley
&Sons
Enzyme Classes: Examples

Class Example Reaction

1 alcohol
O
(oxidoreductase) dehydrogenase
CH3CH2OH + NAD+ CH3CH + NADH + H+

2 hexokinase glucose + ATP  glucose-6-phosphate

(transferase) + ADP

3 chymotrypsin polypeptide + H2O  peptides

(hydrolase)

More examples:Refer Table 5.2, pg 136 , Boyer, R.,

Concepts in Biochemistry, 3rd Ed., 2006, John Wiley
Enzyme Classes: Examples

Class Example Reaction

4 pyruvate OO O
(lyase) decarboxylase CH3C C O + H+ CH3CH + CO2
Removal of group
- nonhydrolysis

5 alanine D-alanine L-alanine

(isomerases) racemase

6 pyruvate
ATP ADP+Pi
(ligases) carboxylase OO
CH3C C O + HCO3-
Forming of
new bond - ATP

_
O OO
O C CH2C C O
Characteristics of enzyme reactions
• What influence the enzyme reaction rate?
1. Substrate concentration
2. Temperature
3. pH
4. Enzyme concentration
5. Inhibitor
Characteristics of enzyme reactions
• Substrate saturation: Increasing the [substrate] increases the
rate of reaction (enzyme activity).
– enzyme saturation limits reaction rates. An enzyme is
saturated when the active sites of all the molecules are
occupied most of the time.
– At the saturation point,
the reaction will not speed
up, no matter how much
additional substrate
is added. The graph of
the reaction rate will plateau.

[substrate] = substrate concentration

Characteristics of enzyme reactions
• Temperature
- very sensitive to temperature changes
- low temp, rate of an enzyme-catalysed reaction increases
proportionally with increasing temperature

Optimal
temperature
Characteristics of enzyme reactions
• Effects of Temperature:
 All enzymes work within a range of
temperature specific to the organism.

 Increases in temperature lead to

increases in reaction rates
– there is a limit to the increase
because higher temperatures lead to
a sharp decrease in reaction rates -
due to the denaturating (alteration) of
protein structure resulting from the
breakdown of the weak ionic and
hydrogen bonding that stabilize the
three dimensional structure of the
enzyme.
Characteristics of enzyme reactions
• pH
- enzymes have an optimal pH at which they function properly
- varies to each other but most in the range of pH 6-8

Optimal pH

pepsin in the stomach works best at a pH of 2

and trypsin at a pH of 8.
Characteristics of enzyme reactions

• Effects of pH: Most enzymes are sensitive to pH and

have specific ranges of activity.
 All have an optimum pH. The pH can stop enzyme
activity by denaturating (altering) the three
dimensional shape of the enzyme by breaking ionic,
and hydrogen bonds.
Characteristics of enzyme reactions
• Enzyme concentration
- the higher the concentration, the greater
should be the initial reaction rate – will be
lasting as long as substrate present

enzyme  enzyme
activity
Characteristics of enzyme reactions
• Inhibitor
- inhibit enzyme by occupy the active site or
bind to other part of enzyme – leading to the
change of enzyme shape and eventually the
active site
- this will decrease the enzymatic reaction rate
Enzyme Inhibitor

http://www.wiley.com/college/pratt/04713
93878/student/animations/enzyme_inhibiti
on/index.html
Enzyme Inhibitors
2 Types of inhibitors:
1. Competitive Inhibitors:
• Compete with substrate for active site
• Occupy the active site so that substare couldn’t bind
there.
• Inhibition can be overcome by adding substrate molecules
to the environment.
2. Non Competitive Inhibitors: 
• Attach to the enzyme in some other place (allosteric site)
than the active site.
• Causes the enzymes active site to change its shape (no
longer complementary to substrate).
• Enzyme can no longer catalyze the reaction.
• Inhibition CANNOT be overcome.
Enzyme Inhibitors
Allosteric Enzymes
• Allosteric enzyme: an oligomer whose biological
activity is affected by other substances binding to it
• these substances change the enzyme’s activity
by altering the conformation(s) of its 4°structure

• Allosteric effector: a substance that modifies the

behavior of an allosteric enzyme; may be an:
1. allosteric inhibitor = negative effectors
2. allosteric activator = positive effectors
Allosteric Enzymes (Cont’d)

• The key to allosteric behavior is the existence of multiple

forms for the 4°structure of the enzyme
• allosteric effector: a substance that modifies the 4°
structure of an allosteric enzyme
• homotropic effects: allosteric interactions that occur
when several identical molecules are bound to the
protein; e.g., the binding of aspartate to ATCase
• heterotropic effects: allosteric interactions that occur
when different substances are bound to the protein;
e.g., inhibition of ATCase by CTP and activation by
ATP
ATCase = aspartate transcarbomylase
CTP = cytidine triphosphate
Allosteric enzymes
• A change in conformational structure at one location of a
multisubunit protein that causes a conformational change at
another location on the protein
• Effectors – i) serves as stimulants to enzyme (+ve effectors)
= increase catalytic activity
– ii) inhibitors (-ve effectors) to enzyme =
reduce/inhibit catalytic activity
- Act by reversible, noncovalent binding to a site on the
enzyme
• Larger and more complex than nonallosteric enzyme
• Have 2 or more subunits (oligomeric)
• Allosteric enzymes have regulatory sites for binding of
substrates and reaction (catalytic sites)
HOMOTROPIC ALLOSTERISM
• eg. Tetrameric allosteric enzyme
composed of 4 identical subunits
• Each subunit has a catalytic site
where substrate/effector will bound
and transformed to product
• Once bound to active site, a
message will be transmitted via
conformational changes to an active
site on another subunit which
makes it easier for a substrate
molecule to bind and react at that
site
• This type (substrate and effector)
are the same is called cooperative
or homotropic
HETEROTROPIC ALLOSTERISM
• A dimer with nonidentical subunits
• Subunit α contain the active site –
catalytic subunit
• Subunit β contains the site for
effector binding – regulatory
subunit
• Binding of a specific effector
molecule to the regulatory site on
the β subunit sends a signal via
conformational changes to the
catalytic site on subunit α
• Substrate and effector different
kinds of molecules - heterotropic
Allosteric enzyme in feedback Inhibition

Formation of
product inhibits its
continued
production –
feedback inhibition
Isoenzymes
• Enzymes that catalyze the same reaction
(catalytically and structurally similar) but are
encoded by different genes

– eg. Glycogen phosphorylase - synthesize in

liver, brain and muscle - involves in
degradation of glycogen

– Isoenzymes = isoforms
Multienzymes
• A group of noncovalently
associated enzymes that
catalyze 2 or more sequential
steps in metabolic/biochemical
pathway
– eg. Glycolysis involve
multienzyme
End of Chapter Enzyme