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ENZYME-

BIOLOGICAL
CATALYST

Key words for today


Enzyme definition
Enzyme catalysis features
Active site, substrate, ES complex

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Catalysis

Structure

Defence

Transport and storage

Regulation

Movement

H2 O2

H2 O +

O2

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

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

Product

Substrate a
reactant in an
enzyme-catalyzed
reaction

Enzyme-substrate (ES) complex the intermediate formed


when the substrate is bind at the active site of an enzyme

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*, ESES*

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

4th step: Release of product EP

E + P
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 strength

Enzyme Catalysis
The rate of a reaction depends on
its activation energy, DG
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?


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.

ES complex

Enzyme Catalysis - Example


Consider the reaction
catalase

H2 O2

H2 O +

O2

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

a/e for the


reaction in the
absence of a catalyst
(a)

(b) a/e lowered in the


presence of an iron
catalyst

(c) energy diagram


for the catalasecatalyzed breakdown
of H2O2

a/e activation energy

(d) energy diagram


for the noncatalysed
breakdown of H2O2 at
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/animations/
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

1
(oxidoreductase)

alcohol
dehydrogenase

2
(transferase)

hexokinase

3
(hydrolase)

chymotrypsin

Reaction
O
CH3CH2OH + NAD+

CH3CH + NADH + H+

glucose + ATP glucose-6-phosphate


+ ADP

polypeptide + H2O peptides

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


Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

Enzyme Classes: Examples


Class

Example

4
(lyase)

pyruvate
decarboxylase

Removal of group
- nonhydrolysis

5
(isomerases)

alanine
racemase

6
(ligases)

pyruvate
carboxylase

Forming of
new bond - ATP

Reaction
O
CH3CH + CO2

OO
CH3C C O + H+
D-alanine

L-alanine

OO
CH3C C O + HCO3_

ATP

ADP+Pi

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/0471393878/stud
ent/animations/enzyme_inhibition/index.html

Enzyme Inhibitors
2 Types of inhibitors:
1. Competitive Inhibitors:
Compete with substrate for active site
Occupy the active site so that substare couldnt 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 enzymes activity
by altering the conformation(s) of its 4structure

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 (Contd)


The key to allosteric behavior is the existence of multiple
forms for the 4structure 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 contain the active site
catalytic subunit
Subunit contains the site for
effector binding regulatory
subunit
Binding of a specific effector
molecule to the regulatory site on
the subunit sends a signal via
conformational changes to the
catalytic site on subunit
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