ENZYMES

INTRODUCTION

Enzymes are biological catalysts. They act by lowering the energy of activation, E a,
without affecting the ΔG of reaction. They are soluble, colloidal catalysts produced by
living cells. They are responsible for carrying out complex reactions rapidly and have
molecular weights ranging from about 12,000 to over a million.

LEARNING OUTCOMES
At the end of this lesson, the student should be able to:
 Describe the chemical nature of enzymes
 Describe the six classes of enzymes based on the type of chemical reactions they
catalyze
 Provide the systematic and common name of an enzyme given a chemical
reaction
 Discuss the factors that affect enzyme activity
 Illustrate the Michelis-Menten equation
 Derive the linear equation of Lineweaver-Burke Plot from the Michelis-Menten
equation
 Discuss the theories behind stereospecificity of enzyme action
 Differentiate the type of enzyme inhibitions
 Illustrate the composition of most enzymes with more emphasis on the
apoproteins and the coenzymes.
 Name the coenzymes and their sources
 Describe how enzyme inhibitions can be used to treat diseases such as Acquired
Immunodeficiency Syndrome (AIDS)

Enzymes are biological catalysts that speed up the rate of the biochemical reaction. Most
enzymes are three dimensional globular proteins (tertiary and quaternary structure).
Some special RNA species also act as enzymes and are called Ribozymes.

Characteristics of Enzymes

 Enzymes speed up the reaction by lowering the activation energy of the reaction.
 Their presence does not affect the nature and properties of end product.
 They are highly specific in their action that is each enzyme can catalyze one kind
of substrate.
 Small amount of enzymes can accelerate chemical reactions.
 Enzymes are sensitive to change in pH, temperature and substrate concentration.
 Turnover number is defined as the number of substrate molecules transformed
per minute by one enzyme molecule.

Intracellular enzymes are synthesized and retained in the cell for the use of cell itself.

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 They are found in the cytoplasm, nucleus, mitochondria and chloroplast.
Example : Oxydoreductase catalyses biological oxidation. Enzymes involved in
reduction in the mitochondria.
Extracellular enzymes are synthesized in the cell but secreted from the cell to work
externally.
Example : Digestive enzyme produced by the pancreas, are not used by the cells
in the pancreas but are transported to the duodenum.

STRUCTURE OF ENZYMES

The active site of an enzyme is the region that binds substrates, co-factors and prosthetic
groups and contains residue that helps to hold the substrate. Active sites generally
occupy less than 5% of the total surface area of enzyme. It has specific shape due to
tertiary structure of protein. A change in the shape of protein affects the shape of active
site and function of the enzyme.

Active site can be further divided into:

Active Site
SiteActive
Binding Site Catalytic Site
It chooses the substrate It performs the catalytic
and binds it to active site. action of enzyme.

Sites of Enzyme Synthesis

Enzymes are synthesized by ribosomes which are attached to the rough endoplasmic
reticulum.
 Information for the synthesis of enzyme is carried by DNA.
 Amino acids are bonded together to form specific enzyme according to the DNA‟s
codes.

The Catalytic Cycle of an Enzyme

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ACTIVATION
Activation is defined as the conversion of an inactive form of an enzyme to active form
which processes the metabolic activity.

TYPES OFACTIVATION
 Activation by co-factors. Many enzymes are activated by co-factors.
Examples: DNA polymerase is a holoenzyme that catalyzes the polymerization of
de-oxyribonucleotide into a DNA strand. It uses Mg- ion for catalytic activity.
Horse liver dehydrogenase uses Zn- ion for its activation.
 Conversion of an enzyme precursor. Specific proteolysis is a common method
of activating enzymes and other proteins in biological system.
Example: The generation of trypsin from trypsinogen leads to the activation of
other zymogens.

Co-factor is the nonprotein molecule which carries out chemical reactions that cannot be
performed by standard 20 amino acids.
Co-factors are of two types:
 Organic co-factors These are the organic molecules required for the proper
activity of enzymes. Example: Glycogen phosphorylase requires the small organic
molecule pyridoxal phosphate.

Types of Organic Co-factors:

1. Prosthetic is a tightly bound 2. Coenzyme is loosely bound organic
organic cofactor e.g. Flavins, co-factor. E.g. NAD
heme groups and biotin.

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  Inorganic cofactors These are the inorganic molecules required for the proper
activity of enzymes. Examples: Enzyme carbonic anhydrase requires Zn for it’s
activity. Hexokinase has co-factor Mg++

An enzyme with its co-factor removed is designated as apoenzyme.

The complete complex of a protein with all necessary small organic molecules, metal ions
and other components is termed as holoenzyme or holoprotein.

The reactant in biochemical reaction is termed as substrate. When a substrate binds to

an enzyme it forms an enzyme substrate complex.

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Nomenclature of Enzymes

 An enzyme is named according to the name of the substrate it catalyses.

 Some enzymes were named before a systematic way of naming enzyme was
formed. Example: pepsin, trypsin and rennin
 By adding suffix -ase at the end of the name of the substrate, enzymes are
named.
 Enzyme for catalyzing the hydrolysis is termed as hydrolase.
Example: maltose + water maltase glucose + glucose

There are Six Major Classes

A systematic classification of enzymes has been developed by International Enzyme

Commission. This classification is based on the type of reactions catalyzed by enzymes.

Main Class of Common Names of

Enzyme Type of Reaction Catalyzed Enzymes
Oxido-reductases Oxidation-reduction Dehydrogenase
Transferases Transfer of an intact group of atoms Transaminase, kinase,
from a donor (usually a coenzyme) to transketolase
an acceptor molecule
Hydrolases Hydrolytic cleavage of chemical bonds Amylase, phosphatase,
such as C-O, C-N, etc. lipase, peptidase
Lyases Non-hydrolytic addition or removal of Decarboxylase,
groups from substrate fumarase, aldolase
Isomerases Any isomerization or intramolecular Isomerase, epimerase,
arrangement mutase
Ligases Joining or condensation of two Carboxylase, thiolase,
molecules coupled with the breaking synthetase
of pyrophosphate bond in ATP or
other nucleoside triphosphate

Example:
Substrate Enzyme Product
Lactose Lactase Glucose + galactose
Maltose Maltase Glucose
Cellulose Cellulase Glucose
Lipid Lipase Glycerol + Fatty acid
Starch Amylase Maltose
Protein Protease Peptides + polypeptide

MECHANISM OF ENZYME ACTION

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The catalytic efficiency of enzymes is explained by two perspectives:
 Thermodynamic changes
All chemical reactions have energy barriers between reactants and products.
The difference in transitional state and substrate is called activational barrier
Only a few substances cross the activation barrier and change into products. That
is why rate of uncatalyzed reactions is much slow. Enzymes provide an alternate
pathway for conversion of substrate into products. Enzymes accelerate reaction
rates by forming transitional state having low activational energy. Hence, the
reaction rate is increased many folds in the presence of enzymes. The total
energy of the system remains the same and equilibrium state is not disturbed.

 Processes at the active site

o Covalent catalysis
Enzymes form covalent linkages with substrate forming transient enzyme-
substrate complex with very low activation energy. Enzyme is released
unaltered after completion of reaction.

o Acid-base catalysis
Mostly undertaken by oxido- reductases enzyme.
Mostly at the active site, histidine is present which act as both proton
donor and proton acceptor.

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 o Catalysis by strain
Mostly undertaken by lyases. The enzyme-substrate binding causes
reorientation of the structure of site due to in a strain condition. Thus
transitional state is required and here bond is unstable and eventually
broken. In this way bond between substrate is broken and converted into
products.

o Catalysis by proximity
In this catalysis molecules must come in bond forming distance.
When enzyme binds: A region of high substrate concentration is produced
at active site. This will orient substrate molecules especially in a position
ideal for them.

Enzyme Kinetics

 Kinetic analysis reveals the number and order of the individual steps by which
enzymes transform substrate into products
 Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of
that enzyme, its role in metabolism, how its activity is controlled, and how a drug
or an agonist might inhibit the enzyme

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 RATES OF REACTION AND THEIR DEPENDENCE ON ACTIVATION ENERGY
 Activation Energy (Ea): “The least amount of energy needed for a chemical
reaction to take place.”
 Enzyme (as a catalyst) acts on substrate in such a way that they lower the
activation energy by changing the route of the reaction.
 The reduction of activation energy (Ea) increases the amount of reactant
molecules that achieve a sufficient level of energy, so that they reach the
activation energy and form the product.
 Example: Carbonic anhydrase catalyses the hydration of 10⁶ CO₂ molecules
per second which is 10⁷x faster than spontaneous hydration.

Factors that Affect the Rate of Enzyme Action

 Substrate concentration [S]. at constant enzyme concentration, [E], increasing
[S] shows a very rapid rise in the initial velocity but a point is reached when no
further increase in velocity is observed.
 Enzyme Concentration [E]. The rate of an enzyme catalysed reaction is directly
proportional to enzyme concentration [E], assuming that [S] is saturated.
 Temperature and pH. As temperature and pH increases, thermal denaturation
decreases the effective [E] and decreases the reaction rate. Most enzymes start
to denature at 450C and optimum pH values range from 4 to 8 (trypsin pH= 8.2)

Rate Constants and Reaction Order

Rate constant (k) measures hoe rapid a reaction occurs

Rate (v, velocity) = (rate constant) (concentration of reactants

v = k1[A]
1st order reaction (rate dependent on concentration of 1 reactant)
v= k-1[B][C]
2nd order reaction (rate dependent on concentration of 2 reactants)
Zero order reaction (rate dependent of reactant concentration)

Michaelis-Menten Model
“According to this model the enzyme reversibly combines with substrate to form an ES
complex that subsequently yields product, regenerating the free enzyme.”

where: S is the substrate, E is the enzyme, ES-is the enzyme substrate complex, P is the
product, K1,K-1 and K2 are rate constants

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Initial Velocity Assumption, measurements made to measure initial velocity (v0). At v0
very little product formed. Therefore, the rate at which E + P react to form ES is
negligible and k-2 is 0. Therefore

Steady State Assumption = [ES] is constant. The rate of ES formation equals the rate of
ES breakdown.

Therefore………if the rate of ES formation equals the rate of ES breakdown

1) k1[E][S] = [ES](k-1+ k2)
2) (k-1+ k2) / k1 = [E][S] / [ES]
3) (k-1+ k2) / k1 = Km (Michaelis constant)

What is Km?
 Km = [S] at ½ Vmax
 Km is a combination of rate constants describing the formation and breakdown of
the ES complex
 Km is usually a little higher than the physiological [S]
 Km represents the amount of substrate required to bind ½ of the available
enzyme (binding constant of the enzyme for substrate)
 Km can be used to evaluate the specificity of an enzyme for a substrate (if obeys
M-M)
 Small Km means tight binding; high Km means weak binding

What is K-cat?
 kcat is the 1st order rate constant describing
 Also known as the turnover # because it describes the number of rxns a molecule
of enzyme can catalyze per second under optimal condition.
 Most enzyme have kcat values between 102 and 103 s-1
 For simple reactions k2 = kcat, for multistep rxns kcat = rate limiting step

What does kcat/Km mean?

 It measures how the enzyme performs when S is low
kcat/Km describes an enzymes preference for different substrates = specificity
constant
 The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse
together (108 to 109 m-1 s-1)
 Catalytic perfection when kcat/Km = diffusion rate
 More physiological than kcat

Limitations of M-M

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  Some enzyme catalyzed reactions that show more complex behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
 Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2
Must optimize one substrate then calculate kinetic parameters for the other
 Assumes k-2 = 0
 Assume steady state conditions

Lineweaver-Burke

Theories on the Formation of the Enzyme-Substrate Complex

Lock and Key Model

Proposed by EMIL FISCHER in 1894. Lock and key hypothesis assume the active site of
an enzymes are rigid in its shape. There is no change in the active site before and after a
chemical reaction. This theory accounts for the absolute specificity of some enzymes.

Induced Fit Model

Proposed by DANIEL KOSH LAND in 1958. The shape of the active site is flexible and
can be induced to fit several structurally similar compounds. Entrance or initial binding
interaction of the substrate induces alterations in the enzymes structure to produce
correct conformation and optimum orientation of the amino residues at the active sites.

Enzyme Specificity
It refers to the presence of an enzyme displays toward certain substrates due to the
uniqueness of the active sites of the enzymes. The uniqueness of the active site depends

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on a) chemical nature, b) electrical charge, and c) spatial arrangement of groups in the
active site.
1. BOND SPECIFICITY
In this type, enzyme acts on substrates that are similar in structure and contain
the same type of bond.
Example: Amylase which acts on α-1-4 glycosidic bond in starch dextrin and
glycogen, shows bond specificity
2. GROUP SPECIFICITY
In this type of specificity, the enzyme is specific not only to the type of bond but
also to the structure surrounding it.
Example:Pepsin is an endopeptidase enzyme, that hydrolyzes central peptide
bonds in which the amino group belongs to aromatic amino acids e. g phenyl
alanine, tyrosine and tryptophan
3. SUBSTRATE SPECIFICITY
In this type of specificity, the enzymes act only on one substrate
Example: Uricase ,which acts only on uric acid, shows substrate specificity.
Maltase , which acts only on maltose, shows substrate specificity.
4. OPTICAL / STEREO-SPECIFICITY
In this type of specificity , the enzyme is not specific to substrate but also to its
optical configuration Example: D amino acid oxidase acts only on D amino acids.
L amino acid oxidase acts only on L amino acids.
5. DUAL SPECIFICITY
 There are two types of dual specificity. The enzyme may act on one substrate by
two different reaction types. Example: Isocitrate dehydrogenase enzyme acts on
isocitrate (one substrate) by oxidation followed by decarboxylation(two different
reaction types) .
The enzyme may act on two substrates by one reaction type
Example: • Xanthine oxidase enzyme acts on xanthine and hypoxanthine(two
substrates) by oxidation (one reaction type)

INHIBITION

Is the prevention of an enzyme process as a result of interaction of inhibitors with the

enzyme. Substances that can diminish the velocity of an enzyme catalyzed reaction is
called an inhibitor. These are compounds capable of combining with the enzyme or the
ES complex, blocking catalysis by the enzyme, it can be specific (inhibits only one
enzyme) or non-specific (which inhibit many enzymes)

TYPES OF INHIBITION

REVERSIBLE INHIBITION. It is an inhibition of enzyme activity in which the inhibiting

molecular entity can associate and dissociate from the proteins binding site.

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1. Competitive inhibition. Inhibitor have close resemblance t the substrate and
competes with the substrate for the active site of the enzyme. Formation of E.S
complex is reduced while a new E.I complex is formed.
Example: Statin drugs such as lipitor compete with HMG-CoA(substrate) and
inhibit the active site of HMG CoA-REDUCTASE (that bring about the catalysis of
cholesterol synthesis).

2. Noncompetitive inhibition. Bind to another part of an enzyme, changing the

function

3. Uncompetitive inhibition.In this type of inhibition, inhibitor does not compete

with the substrate for the active site of enzyme instead it binds to another site
known as allosteric site.
Example: Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are uncompetitive
inhibitors of liver alcohaldehydrogenase

4. Mixed inhibition. In this type of inhibition both E.I and E.S.I complexes are
formed. Both complexes are catalytically inactive.

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IRREVERSIBLE INHIBITION
This type of inhibition involves the covalent attachment of the inhibitor to the enzyme. The
catalytic activity of enzyme is completely lost. It can only be restored only by synthesizing
molecules.
Examples: Aspirin which targets and covalently modifies a key enzyme involved in
inflammation is an irreversible inhibitor.
SUICIDE INHIBITION : It is an unusual type of irreversible inhibition where the enzyme
converts the inhibitor into a reactive form in its active site.

Regulation of enzyme activity helps control metabolism

 A cell’s metabolic pathways must be tightly regulated. Allosteric regulation is the

term used to describe any case in which a protein’s function at one site is affected
by binding of a regulatory molecule at another site.
 They change shape when regulatory molecules bind to specific sites, affecting
function.

Cooperativity. Is a form of allosteric regulation that can amplify enzyme activity

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Feedback Inhibition or feedback control. It is usually a regulatory enzyme or an
allosteric enzyme. It is regulatory in nature because as soon as the product formed is
already in excess of what is desired, the excess product will inhibit an enzyme early in the
sequence of its formation. The end product of a metabolic pathway shuts down the
pathway.

Making It Real

References:

Boyer, R. Concepts in Biochemistry. 3rd Edition.

Campbell, M.K.and Farrell, S.O. (2014) Biochemistry. 8th ed. Brooks Cole.
Malone, L. and Dolter T. (2010). Basic Concepts of Chemistry. 8th edition. Hoboken NJ:
John Wiley & Sons
Mcmurray, J. and Simanek, E. (2008). Fundamentals of Organic Chemistry. Singapore:
Thomson Learning Asia.
Nucum, Z. (2005). Biochemistry for Nursing Students. C & E Publishing Inc.
Sackheim, G. & Lehman, D. (2002). Chemistry for Health Sciences. Pearson Edu. Asia
Pte. Ltd.

Web Pages

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http://www.chemplace.com/college
http://www.yrbe.edu.oncamdhs~science/chemistry
http://www.rsc.org/publishing/journals/gc/about.asp
https://en.m.wikipedia.org
https://opentexttbc.ca
https://www.ncbi.nlm.nih.gov
https://go.roguecc.edu
https://ww2.chemistry.gatech.edu
https://accessmedicine.mhmedical.com
https://byjus.com
https://www.britannica.com
https://www.medicalnewstoday.com
https://www.khanacademy.org

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