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ENZYMES

What are ENZYMES?

macromolecular biological catalysts

accelerate or catalyze chemical reactions

catalyze more than 5,000 biochemical reaction types

Mostly proteins although a few are catalytic RNA


molecules.

increase the rate of a reaction by lowering its activation


energy

SUBSTRATE PRODUCT

They are not consumed in chemical reactions,nor do they


alter the equilibrium of a reaction.
They differ from most other catalysts by being much
more specific.
Enzyme activity can be affected by other molecules:
Inhibitors - molecules that decrease enzyme activity
ex. Drugs & poison
Activators - molecules that increase activity.
Some enzymes are used commercially:
synthesis of antibiotics
household products
biological washing powders
meat tenderizer

"Lock and key" model


To explain the observed specificity of enzymes, in 1894
Emil Fischer proposed that both the enzyme and the
substrate possess specific complementary geometric
shapes that fit exactly into one another.
Induced fit model
In 1958, Daniel Koshland suggested a modification to
the lock and key model: the substrate does not
simply bind to a rigid active site; the amino acid sidechains that make up the active site are molded into
the precise positions that enable the enzyme to
perform its catalytic function.
Induced fit may enhance the fidelity of molecular
recognition in the presence of competition and noise
via the conformational proofreading mechanism.

ETYMOLOGY and HISTORY

(1833) - AnselmePayen

diastase

Louis Pasteur

ferments

(1877) - Wilhelm Khne

term enzyme - comes from Greek "leavened", to describe


this process

enzyme - refer to nonliving substances such as pepsin

ferment - refer to chemical activity produced by living


organisms

(1897) - Eduard Buchner


Zymase
discovered of cell-free fermentation(Nobel Prize in
Chemistry)
enzymes are usually named according to the reaction
they carry out:
the suffix -aseis combined with the name of the
substrate or to the type of reaction

(1926) - James B. Sumner


Urease
catalase (1937)

(1930) - John Howard Northrop & Wendell Meredith


Stanley

Pepsin

Trypsin

Chymotrypsin

Nobel Prize in Chemistry(1946)

(1965) - David Chilton Phillips

x-ray crystallography

Lysozyme

Structural biology
STRUCTURE

globular proteins, acting alone or in larger complexes

linear chains of amino acids that fold to produce a threedimensional structure

larger than their substrates

Sizes range from just 62 amino acid residues to over


2,500 residues in the animal fatty acid synthase.

24 amino acids (catalytic site)


located next to one or more binding sites
allosteric sites where the binding of a small molecule
causes a conformational change that increases or
decreases activity.

MECHANISM
Substrate binding

Enzymes are usually very specific as to what substrates


they bind and then the chemical reaction catalyzed.

complementary shape charge

hydrophilic/hydrophobic characteristics
Enzymes can therefore distinguish between very similar
substrate
molecules
to
be
chemoselective,
regioselective and stereospecific.
Some of these enzymes have "proof-reading"
mechanisms.

an enzyme catalyzes a reaction in a first step

then checks that the product is correct in a


second step
Similar proofreading mechanisms are also found in RNA
polymerase,
aminoacyltRNAsynthetases
and
ribosomes.

Catalysis
Enzymes can accelerate reactions in several ways, all of
which lower the activation energy (G, Gibbs free energy)
1. By stabilizing the transition state:
Creating an environment with a charge distribution
complementary to that of the transition state to
lower its energy.
2. By providing an alternative reaction pathway:
Temporarily reacting with the substrate, forming a
covalent intermediate to provide a lower energy
transition state.
3. By destabilizing the substrate ground state:
Distorting bound substrate(s) into their transition state
form to reduce the energy required to reach the
transition state.
By orienting the substrates into a productive
arrangement to reduce the reaction entropy change.
The contribution of this mechanism to catalysis is
relatively small.

Dynamics
Enzymes are not rigid, static structures; instead they have
complex internal dynamic motions that is, movements of
parts of the enzyme's structure such as individual amino
acid residues, groups of residues forming a protein loop or
unit of secondary structure, or even an entire protein
domain.
Allosteric modulation
Allosteric sites are pockets on the enzyme, distinct from the
active site, that bind to molecules in the cellular
environment.

These molecules then cause a change in the


conformation or dynamics of the enzyme that is
transduced to the active site and thus affects the
reaction rate of the enzyme. In this way, allosteric
interactions can either inhibit or activate enzymes.
COFACTORS

non-protein molecules required to be bound for activity


Inorganic cofactors
Organic cofactors

Coenzymes - which are released from the


enzyme's active site during the reaction

Prosthetic groups - which are tightly bound to an


enzyme

Ex. carbonic anhydrase

apoenzymesor apoproteins
Enzymes that require a cofactor but do not have one
bound

Holoenzyme

An enzyme together with the cofactor(s) required for


activity

It can also be applied to enzymes that contain multiple


protein subunits

Coenzymes

They are small organic molecules that can be loosely or


tightly bound to an enzyme.

They transport chemical groups from one enzyme to


another.

Since they are chemically changed as a consequence of


enzyme action, it is useful to consider coenzymes to be a
special class of substrates, or second substrates, which
are common to many different enzymes.

They are usually continuously regenerated and their


concentrations maintained at a steady level inside the
cell.
THERMODYNAMICS

Enzymes increase reaction rates by lowering the energy of


the transition state.
First, binding forms a low energy enzyme-substrate
complex (ES).
Secondly the enzyme stabilizes the transition state
such that it requires less energy to achieve compared
to the uncatalyzed reaction (ES).
Finally the enzyme-product complex (EP) dissociates to
release the products.
KINETICS
Enzyme kinetics is the investigation of how enzymes bind
substrates and turn them into products which are commonly
obtained from enzyme assays.
MichaelisMenten kinetics
A quantitative theory of kinetic enzyme proposed in
1913 by Leonor Michaelisand Maud Leonora Menten.
In the first, the substrate binds reversibly to the
enzyme, forming the enzyme-substrate complex. The
enzyme then catalyzes the chemical step in the
reaction and releases the product.
This work was further developed by G. E. Briggs and J.
B. S. Haldane, who derived kinetic equations that are
still widely used today.

INHIBITION

Types of Enzyme Inhibitors

Competitive

They are strongly resemble the real substrate of the


enzyme.

This type of inhibition can be overcome with high


substrate concentration.

In some cases, the inhibitor can bind to a site other than


the binding-site of the usual substrate and exert an
allosteric effect to change the shape of the usual
binding-site.

Non-competitive

A non-competitive inhibitor binds to a site other than


where the substrate binds.

In contrast to competitive inhibition, non-competitive


inhibition cannot be overcome with high substrate
concentration.

Uncompetitive

An uncompetitive inhibitor cannot bind to the free


enzyme, only to the enzyme-substrate complex; hence,
these types of inhibitors are most effective at high
substrate concentration.

In the presence of the inhibitor, the enzyme-substrate


complex is inactive.

This type of inhibition is rare.


Mixed

A mixed inhibitor binds to an allosteric site and the


binding of the substrate and the inhibitor affect each
other.

This type of inhibitor does not follow the MichaelisMenten equation.

Irreversible

An irreversible inhibitor permanently inactivates the


enzyme, usually by forming a covalent bond to the
protein.
Ex: Penicillin and aspirin
Functions of inhibitors

In many organisms, inhibitors may act as part of a


feedback mechanism. If an enzyme produces too much
of one substance in the organism, that substance may
act as an inhibitor for the enzyme at the beginning of the
pathway that produces it, causing production of the
substance to slow down or stop when there is sufficient
amount.
ENZYMES
CONTENTS
Biological Function
Metabolism
Control of Activity
Involvement in Disease
Naming Conventions

Industrial Applications

Cleavage of the polypeptide chain


Chymotrypsin, a digestive protease, is produced in
inactive form as chymotrypsinogen in the pancreas and
transported in this form to the stomach where it is
activated. This stops the enzyme from digesting the
pancreas or other tissues before it enters the guts. This
type of inactive precursor to an enzyme is known as
zymogen.
Note:For
example,
in
the
response
to insulin,
the phosphorylation of
multiple
enzymes,
including glycogen synthase, helps control the synthesis
or degradation of glycogen and allows the cell to respond
to changes in blood sugar.

BIOLOGICAL FUNCTION
ENZYMES

are indispensable for signal transduction and cell


regulation (kinases, phosphatases)

generate movement, with myosin hydrolyzing ATP to


generate muscle contraction

transport cargo around the cell as part of the


cytoskeleton
An important function of enzyme is in the digestive
systems of animals. Enzymes such as amylases and
proteases break down large molecules into smaller ones so
that they can be absorbed by the intestines.
Different enzymes absorbed different food substances.

3. Quantity
Enzyme production can be enhanced or diminished by a
cell in response to changes in the cells environment.
This form of gene regulation is called induction.
Note: Starch molecules, for example, are too large to be
absorbed from the intestine, but enzymes hydrolyze the

Enzymes in the liver called cytochrome P450


starch chains into smaller molecules such as maltose and
oxidases, which are important in drug metabolism.
eventually glucose, which can then be absorbed.
Induction or inhibition of these enzymes can
cause drug interactions.
METABOLISM

Enzyme levels can also be regulated by changing the rate


of enzyme degradation.

Several enzymes can work together in a specific order,


creating metabolic pathways.
4. Subcellular Distribution
One enzyme takes the product of another enzyme
Enzymes can be compartmentalized with different
as a substrate. After catalytic reaction, the product
metabolic pathways occurring in the different cellular
is then passed on to another enzyme.
compartments.
Note: Sometimes more than one enzyme can catalyze the
Trafficking of enzyme to different compartment may
same reaction in parallel; this can allow more complex
change the degree of protonation or oxidative state
regulation: with, for example, a low constant activity
which in turn affects enzyme activity.
provided by one enzyme but an inducible high activity from a
Note: For example, fatty acids are synthesized by one set of
second enzyme.
enzymes
in
the cytosol, endoplasmic

Enzymes determine what steps occur in


reticulum and golgi and used by a different set of
these pathways. Without these, metabolism would
enzymes as a source of energy in the mitochondrion,
neither progress through the same steps and could not
through -oxidation.
be regulated to serve the needs of the cell.

Note: Most central metabolic pathways are


regulated at a few key steps, typically through enzymes
whose activity involves the hydrolysis of ATP. Because
this reaction releases so much energy, other reactions
that are thermodynamically unfavorable can be coupled
to ATP hydrolysis, driving the overall series of linked
metabolic reactions.

The metabolic pathway of glycolysis releases energy


by converting glucose to pyruvate by via a series of
intermediate metabolites. Each chemical modification (red
box) is performed by a different enzyme.
CONTROL OF ACTIVITY
5 Main Ways that Enzyme Activity is Controlled

Regulation

Post-translational Modification

Quantity

Subcellular Distribution

Organ Specialization
1. Regulation

Enzymes can either be activated or inhibitedby other


molecules. For example, the end product(s) of a metabolic
pathway are often inhibitors for one of the first enzymes of
the pathway thus regulating the amount of end product
made by the pathways.

Negative feedback mechanism can effectively adjust


the rate of synthesis intermediate metabolites according to
the demands of the cells.
Note: 1. Sucha regulatory mechanism is called a negative
feedback mechanism, because the amount of the end
product produced is regulated by its own concentration.
2. This helps with effective allocations of materials and
energy economy, and it prevents the excess manufacture
of end products.
2. Post-translational Modification

e.g.
phosphorylation,
glycosylation

myristoylationand

5. Organ Specialization
In multicellular eukaryotes, cells in different organs and
tissues have different patterns in gene expression and
therefore have different sets of enzymes available for
metabolic reactions.

Provides a mechanism for regulating the overall


metabolism of organism.
Note:For example, hexokinase, the first enzyme in
the glycolysis pathway,
has
a
specialized
form
called glucokinase expressed
in
the liver and pancreas that has a lower affinity for glucose
yet is more sensitive to glucose concentration. This
enzyme
is
involved
in
sensing blood
sugar and
regulating insulin production.

INVOLVEMENT IN DISEASE

Since the tight control of enzyme activity is essential


for homeostasis, any malfunction of a single critical
enzyme can lead to genetic disorder.
The malfunction of just one type of enzyme out
of the thousands of types present in human body can be
fatal.
An example of a fatal genetic disease due to enzyme
insufficiency is Tay-Sachs disease, in which patients
lack the enzyme hexosaminidase.
One example of enzyme deficiency is the most common
type of phenylketonuria . Many different single amino
acid mutations in the enzyme phenylalanine hydroxylase,
which catalyzes the first step in the degradation of
phenylalanine, result in build-up of phenylalanine and
related products.
Some mutations are in the active site, directly disrupting
binding and catalysis, but many are far from the active
site and reduce activity by destabilising the protein
structure, or affecting correct oligomerisation.This can
lead to intellectual disability if the disease is untreated.

NAMING CONVENTIONS

An enzymes name is often derived from its substrate


or the chemical reaction it catalyzes, with the word
ending in ase.

Different enzymes that catalyze the same reaction are


called isozymes.

The International Union of Biochemistry and


Molecular Biologyhave developed a nomenclature for
enzymes, the EC numbers.
INDUSTRIAL APPLICATIONS
Enzymes are used in the chemical industry and other
industrial applications when extremely specific catalysts
are required. Enzymes in general are limited in the
number of reactions they have evolved to catalyze and
also by their lack of stability in organic solvents and at
high temperatures.

EC
1
EC

Oxidoreducta
ses
Transferases

catalyze
oxidation/reduction
reactions
transfer a functional group

Hydrolases

catalyze the hydrolysis of various


bonds
cleave various bonds by means other
than hydrolysis and oxidation
catalyze
isomerization
changes
within a single molecule

2
EC
3
EC

Lyases
4

EC

Isomerases
5

EC

Ligases

join 2 molecules with covalent bonds

6
Application
Biofuel
Industry

Biological
Detergen
t

Brewing
Industry
Culinary
Uses

Amylase, glucana Split


Enzymes
Uses
ses,
polysaccharid
used
proteases
es
and
Cellulases
Break down cellulose into
proteins in the
sugars that can be
malt.
fermented
to
Betaglucana Improve the wort and beer
produce cellulosic
ses
filtration characteristics.
ethanol.
Amylogluco
Make
low-calorie beer and
Ligninases
Pretreatment
sidase
adjust fermentability.
of biomass for
biofuel
and
production.
pullulanases
Proteases, a Remove protein, starch,
Acetolactate Increase
fermentation
mylases,
and fat or oil stains
decarbox
efficiency by reducing
lipases
from
laundry
and
ylase(AL
diacetyl formation.
dishware.
DC)
Mannanases Remove food stains from
the
common
food
additive guar gum.

Papain

Tenderize meat
cooking.

for

BIOCATALYSIS
Biocatalysts are either proteins (enzymes), or in a few
cases, they may nucleic acids.
Enzymes are necessary in all living systems, to catalyze
all chemical reactions required for their survival and
reproduction rapidly, selectively and efficiently.
Their application covers the production of desired
products for all human material needs, as well as in a
wide
range
of
analytical
purposes,
especially
in
diagnostics.

Biomass can be used as a raw material to meet the human


needs.

Biotechnological processes are specially suited to the


production of compounds from biomass to raw materials.

Biotechnology is the integration of natural sciences and


engineering sciences in order to achieve the application of
organisms, cells, parts thereof and molecular analogues for
products and services.
- European Federation of Biotechnology,
1989
HISTORICAL HIGHLIGHTS OF ENZYME TECHNOLOGY /
APPLIED BIOCATALYSIS
Cheese making has always involved the use of
enzymes, and as far back as about 400 BC, Homers Iliad
mentions the use of a kids stomach for making cheese.
During the 18th and 19th centuries, applied biocatalysis
began to develop a more scientific basis:
1833 Payen and Persoz investigated the actions of
extracts of germinating barley in the hydrolysis of
starch to yield dextrin and sugar.
1874 Christian Hansen started the first company for
marketing of standardized enzyme preparations namely
rennet for cheese making.
1878 Kuhne introduced the name enzymes for those
substances that were previously called ferments.
1894 Emil Hermann Fischer proposed his famous lockand-key model to explain the mechanism of enzymatic
catalysis and its substrate specificity. He also stated the
protein nature of enzymes.
1897 It was demonstrated by Eduard Buchner that
alcoholic fermentation was possible with a cell-free
press juice. This discovery marked the end of the
established paradigm (by L. Pasteur) that living organisms
require a vital force (visvitalis).
1897 Bertrand coined the term coenzymes.

1898 Croft-Hill performed the first enzymatic sysnthesis


of isomaltose by allowing a yeast extract to act on a 40%
glucose solution.
1900 Kastle and Loevenhart showed that the hydrolysis of
fat and other esters by lipases was a reversible
reaction, and that enzymatic synthesis could occur in a
dilute mixture of alcohol and acid.
1907 Otto Rohm patented the enzymatic treatment of
leather with a mixture of trypsin and ammonium salts; in
the same year, the Rohm Company was founded.
1950 Penicillin amidaseused for the hydrolysis of
penicillin was first identified followed some years later by
glucose isomerase, which is used to isomerize glucose to
fructose.
1955 Georg Manecke was one of the first to suceed in
making relatively stable immobilized systems of
proteins on polymer carriers.
1960 Novo Industry (now Novozymes) started a largescale production of proteases from Bacillus licheniformisin
submerge culture.
1972 Introduction of gene technology; the first company
in this field was Cetus Corporation in Berkeley, USA.

Goals and Essential System


Improved Enzyme Processes
GOALS

Properties

for

New

or

Biotechnological Processes:
The Use of Isolated or Intracellular Enzymes as
Biocatalysts
Biotechnological processes use one or more enzymes
with or without cofactors or cosubstrates as biocatalysts. When
more enzymes and cosubstrate regeneration are required,
fermentation processes with living cells are more effective than
processes with isolated enzymes.
A cofactor is a non-protein chemical compound that is
required for the protein's biological activity. These proteins are
commonly enzymes, and cofactors can be considered "helper
molecules" that assist in biochemical transformations.
ADVANTAGES compared with fermentations:
Higher space-time yields can be obtained than with living cells;
smaller reactors can then be used, reducing processing costs.
The risk that a desired product is converted by other enzymes
in the cells can be reduced.
The increased stabilty and reuse of immobilized biocatalysts
allows continuous processing for up to several months.
For such enzyme processes:
1. The required intra- or extracellular enzyme must be
produced in sufficient quantities and purity (free from
other disturbing enzymes and other compounds).
2. Cells without intracellular enzymes that may disturb the
enzyme process must be selected.
3. The enzyme costs must be less than 510 % of the total
product value
Advantage:
Enzymes are proteins that are essential for living
systems and, in the right place, they catalyze all chemical
conversions required for the systems survival and reproduction.
Disadvantage:
However, in the wrong place they can be harmful to an
organism.
Advantage:
Enzymes are normal constituents of food and, as with all
orally ingested proteins, they are hydrolyzed in the stomach and
intestine.
Disadvantage:
However, if enzymes or other proteins are inhaled as
small particles or aerosols in the lungs, they can be transferred
directly into the bloodstream.
An aerosol is
a colloid of
fine solid particles
or liquid droplets, in air or another gas.

Enzyme processes have become competitive and have


been introduced into industry when they attain these goals
better than alternative processes. This, however, also requires
that these goals are quantified such that the amount of product
and byproducts (or waste) produced with a given amount of
enzyme in a given time must be determined.
TWO
CATEGORIES
OF
ENZYME
PROCESSESTWO
CATEGORIES OF ENZYME PROCESSES
1. Equilibrium-controlled
processes:
the
desired
product concentration or property has a maximum at
the end-point of the process the chemical equilibrium is
independent of the properties of the catalyst (enzyme),
but is dependent upon on pH and temperature.
2. Kinetically controlled processes: the desired product
concentration or property (such as fiber length or
smoothness in textiles or paper) reaches a maximum,
the concentration or properties of which depend on the
properties of the catalyst, pH, and temperature. The
process must be stopped when the maximum is
reached.

weight basis less than 1 of all produced enzymes are


used for these applications.
Some enzymes are produced in increasing amounts for
therapeutic purposes.
During the past 10 years, the three- dimensional
structures and detailed mechanisms of the reactions
that they catalyzed have been determined for many of
the enzymes seen to be important in enzyme
technology.

(BIO)CHEMISTS
Determine the mechanism and properties of the
catalyzed processes, the kinetics of the enzymecatalyzed process and other relevant properties
(stability, solubility, pH- and temperature dependence of
equilibrium constants, selectivities).

Essential System Properties for Rational Design of an


Enzyme Process

MICRO- AND MOLECULAR BIOLOGISTS


Provides information about the properties of the
enzymes should be improved (specificity, selectivity, pH
optimum, stability, metal ion requirement, yield in the
fermentation).
(BIO)CHEMICAL ENGINEERS
Scale up the process to the production scale.
They identify problems that must be solved by the
(bio)chemists and micro- and molecular biologists.
Fields where large amounts of enzymes will be required:
The production of optically pure therapeutics and fine
chemicals.
The synthesis of antibiotics
Paper production or recycling to reduce waste and
energy consumption.
The
regioand
stereoselective
synthesis
oligosaccharides for food and pharmaceutical processes
The selective glycosylation of peptides, proteins and
other drugs.
Environmental biotechnology

Current Use and Potential of Enzyme Technology

Besides industrial uses, many enzymes are used for


analytical purposes, mainly in diagnostics, though on a

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