1. Overview of Enzymes
Dr. Anil V Dusane
Sir Parashurambhau College
Pune, India
anildusane@gmail.com
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2. Introduction
• Kuhne (1868) coined term ‘Enzyme’.
• Eduard Buchner named the enzyme that brought about the
fermentation of sucrose, ‘zymase’.
• Enzyme (En = in, Zyme = yeast) is a Greek work that refers to the
occurrence in yeast of something responsible for its fermentative
activity.
• Enzymes are indispensable compounds that play key role in
metabolism by bringing direction and control to the physiological
processes of the living cells.
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3. Definition of enzyme
• Enzymes are also called as Biological catalyst, ‘Biocatalyst’, a
substance (enzyme) that initiates or modifies the rate of a
chemical reaction in a living body.
• Definition: Enzymes are organic substances (simple or
compound proteins) capable of catalyzing reactions in living
systems.
• Enzyme initiates and accelerate biochemical
reactions and lowers activation energy (energy
required to carry out reaction) in the cell.
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4. Nomenclature and classification
• Named according to the substrate they attack (protease, lipase, DNAase
etc.) or the type of reactions they catalyze (oxidases, reductases,
transaminases, etc).
• Enzymes are complemented with suffix ‘ase’.
• International Union of Biochemistry IUB (1972) has recognized six
major classes of enzymes based on reactions they catalyzes.
Rules of IUB for nomenclature and classification of enzymes
• Each enzyme has a systematic code number (E.C.) of four digits.
• First digit indicates main class
• Second digit indicates subclass.
• Third digit indicates subdivision of sub-class(sub-subclass)
• Fourth digit designates the serial number of specific enzyme in the
fourth sub-class. 4

5. Nomenclature
• E.g. E.C.1.1.1.1 stands for enzyme dehydrogenase where
E.C. stands for Enzyme Commission
1. Stands for oxidoreductase,
1.1 for enzyme which utilizes substrate as –CHOH (alcoholic group),
1.1.1 stands for those enzymes which utilizes NAD as acceptor.
• Lactate dehydrogenase is an oxidoreductase is written as
EC 1.1.1.27.
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6. Classification of enzymes- Six classes
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7. Classes of enzymes
1. Oxido-reductases
• Catalyze biological oxidation and reduction
• One compound is oxidized and another reduced.
• Deydrogenases-catalyses removal of 2 atoms of hydrogen
• Oxidases-catalyses the reduction of O2
• Oxygenase-catalyse incorporation of O2 in to substrate
• Peroxidases-use of H2O2 as oxidant
• E.g. dehydrogenases, oxidases, oxygenases, oxidative
deaminases, hydroxylases and peroxidases.
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8. Classes of enzymes
2. Transferases
• Bring about the transfer a group from one molecular to another.
• Catalyses exchange of groups between two substrates AB + CD =
AC + BD.
• aminotransferases catalyze exchange of amino and keto group
between amino and keto acid
• Glutamic acid + OAA ==(transaminase) -glutaric acid +
aspartic acid
• Eg. transaminases, kinases, hexokinases, etc.
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9. Classes of enzymes
3. Hydrolyses
• Catalyses hydrolysis of complex substrates into simpler ones.
• Starch into glucose
• AB + HOH ---- AH+ + BOH- (AB substrate)
• Sucrose + H2O ---------- (sucrase, invertase) glucose + fructose
• E.g. carbohydrases, esterases, proteases, etc
• Catalyses hydrolysis reaction AB + H2O = A.OH + HB. Add H2O and breaking
substrate
i)peptidases-catalyses hydrolysis of peptide bonds
ii)glycosidases- catalyses glucosidic bonds.
iii) deaminases-catalyses hydrolysis of amines
iv)Sucrase- Sucrose + H2O sucrase invertase= Glucose + fructose
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10. Classes of enzymes
4. Lyases
• Result in a direct removal of groups from substrate non-
hydrolytically
• AB = A + B
• Lyases act on C-C-, C-O, C-N, C-S and C-halide bonds.
• In most of the cases coenzyme is required for the activity.
E.g. Decarboxylases, aldolases, dehydratases, etc.
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11. Classes of enzymes
5. Isomerases
• Catalyses isomeric changes (Isomerization) so called as
isomerases.
• Catalyses isomerization of substances (substrates) optical or
structural isomers
• Eg. Isomerase, epimerase
• Glucose-6-phosphate == (phosphohexose isomarase) Fructose-
6-phosphate.
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12. Classes of enzymes
6. Ligases (synthetases)
• Catalyze the joining of two molecules coupled with the
breakdown of a pyrophosphate bond in ATP
• Catalyze synthesis of different types of bonds such as C-N, C-S,
C-O etc.
• Glutamate + NH3+ ATP ------(glutamine synthetase) glutamine +
ADP + Pi
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13. Chemical nature of enzymes
Enzymes are proteinaceous in nature.
Some enzymes also contain a non–protein group
or prosthetic.
Protein part of enzyme is also called apoenzyme
Complete enzyme with prosthetic group is called
as holoenzyme.
Organic prosthetic groups are called coenzymes
while inorganic prosthetic group is called cofactor
Protein part (apoenzyme) + non-protein part
(prosthetic group) = holoenzyme.
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14. Properties of enzymes
1. Specificity
•Each enzyme is specific in the sense bond
specificity, group specificity, substrate
specificity, optical specificity, geometrical
specificity and co-factor specificity.
•Enzymes it can operate only upon certain
substrate or group of substrates.
•Each enzyme act only upon substances having a
certain molecular pattern and can affect only
one particular type of chemical bond only.
•Many enzymes apparently act on only a single
kind of substrate. E.g. urease can act only upon
urea and no other molecules. 14

15. Properties of enzymes
2. Colloidal nature
• Molecules of enzymes are large in size and characterized as the
particles of colloidal systems.
• Colloidal nature provides an extensive surface area for chemical
reactions
3. Proteinaceous in nature
• All enzymes except ribozymes are protein in nature.
• React with both acidic and alkaline substances.
• Soluble in water, salt solution, alcohol and dilute glycerin.
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16. Properties of enzymes
4. Catalytic activity
• There may positive or negative catalysis. However, there is mostly positive catalysis
• Small quantity of enzyme can bring about transformation of vastly large amounts of
the substrate.
• Enzymes can function at very low conc.
• Rate of reaction is directly proportional to enzyme concentration
• Invertase catalyzes the conversion of at least 1,000,000 times its own weight of
sucrose.
5. Thermolabile:
• Enzymes are proteins hence they are thermolabile (sensitive to temperature). At
60-700C enzymes are destroyed. This due to the heat coagulation phenomenon.
• There is always a specific temperature of optimum activity of every enzyme, which
usually ranges from 250C to 450C.
• Enzymatic action is highest at 370C and enzymes.
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17. Properties of enzymes
6. Enzyme inhibitors:
• These are certain product that inhibits enzyme activity
• During reaction, the active sites of the enzymes are filled up with
these inhibitors instead of substrate molecules
• Drugs, antibiotics and poisons inhibit enzyme inhibitors.
7. Reversibility of action:
• Enzymes can accelerate the rate of reaction in whichever direction
it is taking place, provided suitable sources of energy available.
• Usually a single enzyme brings about the synthesis and digestion
(hydrolysis) of a particular substance.
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18. Factors affecting enzyme activity
1. Temperature
• Low temperature inactivates proteins.
• At high temperature protein looses secondary
and tertiary structures.
• Enzyme activity gets doubled at every 10oC.
• Kinetic energy increases as temperature
increases.
• An enzyme shows maximum activity at
optimum temperature 25-30oC.
• However, beyond 60-70oC the enzyme activity
is permanently stopped
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19. Factors affecting enzyme activity
2. pH
• Most of the enzymes are extremely sensitive to
pH
• Wrong pH denatures enzymes, disturbs ionic
state of enzyme and substrate.
• It also affects the binding of prosthetic group
• Optimum pH shows better enzyme activity
• In general pH range 5-9.
• catalase shows optimum activity at 9.0 pH
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20. Factors affecting enzyme activity
3. Substrate concentration
• Increased concentration of substrate brings about an increase
in the activity of enzyme
• Beyond a particular point though we increase the
concentration of substrate there is no change in enzyme
activity
• Active sites of enzymes become saturated; no active sites are
available, so the rate remains same though the concentration
increases.
4. Enzyme concentration
• Rate of reaction follows the increased concentration of
enzyme until there is enough concentration of substrate is
present
• Invertase catalyzes the conversion of at least 100000 times its
own weight of sucrose.
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21. Factors affecting enzyme activity
5. Effects on ions
• Hydrogen ion concentration is the most important factor in activity of
all enzymes.
• Cations like Mg++, Ca++, Na+, Zn++, K+ also play an important role in
activity of certain enzymes
• Enzymes in the absence of particular cation remain inactive.
6. Accumulation of end products
• It retards the rate of reaction due to change in pH of enzyme solution.
• Enzyme become inactive and increase in the rate of reverse reaction
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22. Enzyme inhibitors
• Specific chemicals can inhibit most of enzymes
• Two types of inhibitors irreversible and reversible
• Irreversible inhibitors
• These combine with or destroy a functional group on
enzyme molecule that is necessary for its catalytic
activity
• E.g. Di-iso Propyl Fluorophosphate (DPF) that inhibits
enzyme cholinesterase.
• Irreversible inhibition results from the formation of
stable enzyme inhibitor (EI) complex that results in
complete inhibition of the enzyme.
• E.g. inhibition of Xanthine oxidase by CN-.
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23. Reversible inhibition
• Inhibitors do not cause permanent damage in the
functional groups and once these inhibitors are
removed the enzyme become fully active.
• There are mainly two types viz. competitive
inhibition and non-competitive inhibition.
Competitive inhibitors
• A competitive inhibitor competes with the substrate
for binding to the active site but once bound can not
be transformed by the enzyme.
• These inhibitors usually resemble to normal
substrate in 3D structure and can bind with the
active site of enzyme in the same way as normal
substrate molecule binds.
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24. Reversible inhibition
• The inhibitor molecules can not be attacked by enzyme molecule
and since their active site is occupied, they become non-
functional for normal substrate also.
• Competitive inhibitor increases Michalis constant, but it has no
effect on Vmax.
• Many antibacterial drugs work on principle of competitive
inhibitors of bacterial enzymes.
• Inhibition of succinic dehydrogenase by malic acid.
Ki
• E+I ======= EI where E enzyme, I inhibitor, Ki inhibitor
association complex.
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25. Reversible inhibition
Non-Competitive inhibitors
• Not very specific and they bind at the site on the enzyme other than the
catalytic site.
• It alters the configuration of enzyme molecule so that the reversible
inhibition of enzyme activity occurs.
• Inhibition is not reversed by increasing concentration of substrate.
• Heavy metals and cyanide act as non-competitive inhibitors of enzymes.
• The level of inhibition is controlled by concentration of inhibitor
• Non-competitive inhibitors decrease the Vmax of the enzyme but they
have no effect on Km.
• Eg. Cytochrome oxidase
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26. Allosteric inhibition
• In this type of inhibition the inhibitor which is structurally quite
different from the substrate is bound at a site other than the
active site of enzyme.
• This binding of the inhibitor alters the conformation of the
enzyme proteins and there by prevents it from binding to the
substrate.
• Since the inhibitors bind at a site other than the active site of
the enzyme they are called allosteric effectors or determinant
and the site to which they bind, allosteric sites (allows=other)
• The whole phenomenon is called as allosteric effect or
feedback inhibition and it is always reversible.
• Allosteric inhibition has a great physiological and biochemical
importance.
• Allosteric enzymes are formed by the aggregation of many
subunits
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27. Activators
• Enzyme activators are molecules that bind to enzymes and
increase their activity.
• These are the opposite of enzyme inhibitors. These molecules
are often involved in the allosteric regulation of enzymes in the
control of metabolism.
• Activators like Mg++, Ca++, Mn++ etc. may take part in the
formation of enzyme-substrate complex.
• Mn++ in the action of some peptidases may prevent the
inactivation of the enzyme by inhibitors.
• E.g. kinases - enterokinases converts trypsinogen into trypsin
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28. Coenzymes
• Many reactions of substrate are catalyzed by enzymes
only in the presence of non-protein organic molecule
called coenzyme.
• Coenzyme combines with the apoenzyme (protein part)
to form holoenzyme.
• Coenzymes are small molecular weight organic,
dialyzable, thermostable compounds.
• Required for the catalytic activity of one or more group
of enzymes.
• Co-enzymes are heat-stable non-protein organic
molecules.
• Examples: Nicotinamide Adenine Dinucleotide (NAD),
riboflavin coenzyme, coenzyme-A, lipoic acid, etc.
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29. Coenzymes
Classification based on
Chemical characteristics: ATP, NAD, NADP, FMN.
Functional characteristics: CoA, Thiamine PyroPhosphate (TPP).
Nutritional characteristics: Folic acid coenzyme, B12 coenzyme, Biotin
Functions of coenzymes
• To accept atoms or groups from a substrate and transfer them to other
molecules.
• NAD and NADP coenzyme functions as hydrogen acceptor in dehydrogenation
reactions.
• Main function of CoA is to carry acetyl groups and they are used in oxidative
decarboxylation of pyruvic acid and synthesis of fatty acids and acetylation.
• Pyridoxal phosphate (B6-PO4) is involved in transamination reactions. 29

30. Active sites of enzymes
• It is also known as catalytic activity site
• Some restricted region of the enzymes, which is concerned with
process of catalysis termed as active site.
• One or more regions on the enzyme molecules where the substrate
can bind.
• Shape of the enzyme molecule is such that it will expose some amino
acids so that substrate molecules can bind to it for necessary catalytic
function
• Binding of substrates to the enzyme involves only its active site.
• If the shape of enzyme molecule is altered, the active site is also likely
to be displaced and it hampers the catalytic function.
• If certain enzymes trimmed to smaller sizes they will not loose their
catalytic activity
• E.g. papain may be trimmed to 60 from 180 amino acid residues with
out loosing its activity
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31. Mode of action of enzyme
• Enzyme substrate-complex theory (most accepted) has
been proposed
Enzyme substrate complex theory
• Michaellis and Menton (1913) proposed to explain mode
of enzyme action.
• Enzymes have certain active sites for the attachment of
substrate molecule where an enzyme can form an intimate
relationship with substrate.
• Enzyme forms a weakly bound compound with substrate
which on hydrolysis decomposes into the reaction
products.
• In simple form theory can be represented as follows
• Enzyme + substrate (ES) ===== enzyme substrate complex
(ES) === End products (P) + enzyme (E)
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32. Models for Active site
Rigid model of active site (lock and Key model)
• According to this enzyme and substrate are strictly
complementary structures
• During the complex formation, substrate fits exactly with the
active site of enzyme as a key fits into a lock.
Flexible model of active site (Induced fit model)
• According to this, the active site is not very rigid and its
configuration changes according to the substrate configuration
so that there is an induced fit between enzyme and substrate
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33. Models to explain mode of enzyme action and their specificity
• Two models viz. Lock-key model and Induced fit
model has bee proposed
Lock-key model
• Fischer (1898) proposed this model which was later
advanced by Paul Fields and D.O. Woods.
• According to this model the enzyme-substrate
complex formation is analogous to the fitting of lock
and key.
• During the complex formation, substrate fits exactly
with the active site of the enzyme as a key fits into a
lock.
• As a particular lock can be opened by a particular key
in the same way particular enzyme acts on a
particular substrate
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34. Lock and Key model
• This theory depends on physical contact between substrate and enzyme
molecules.
• It is least accepted model as compared to induced fit model.
• This model is rigid, and enzymes do not complimentary to the substrate.
• Catalytic sites are fixed and there is no proper orientation of the active sites
• There is unchangeable configuration of enzymes.
• This theory is supported from the study of competitive inhibition
• Competitive inhibitors have some structural similarity with the substrate
molecule, both of which compete for the same active site on the enzyme.
• If some part of the active site is preoccupied by competitive inhibitors, the
substrate will not be able to combine with it. Thus the activity of enzyme is
inhibited like a wrong key can not open a lock.
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35. Induced fit model
• Koshland (1959) proposed this model
• According to this model the attachment of
the substrate to the active sites brings about
a change in 3D structure of the enzyme.
• This results in the precise orientation of the
catalytic groups in the enzyme molecule
which causes the enzyme reaction.
• Enzyme changes shape upon binding with
the substrate, when its active sites assume a
shape complementary to that of the
substrate.
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36. Induced fit model
• According to this theory the active centers of the substrate and
the enzyme fit into each other and they combine to form an
active complex
• Studies of optical rotation measurements and X-ray diffraction
analysis of several enzymatic reactions support this theory
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37. Questions
• What are enzymes? How are they classified? Describe its mode of action.
• What are enzymes? Write an account of the factors controlling enzymatic reactions.
• What are enzymes? Give an account of the general properties and nomenclature of
enzymes.
• Short notes
i) Lock-key model
ii) Ligases
iii) Induced fit theory
iv) Active sites
v) Competitive inhibitors
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38. Thanks
Dr. Anil Dusane
Sir Parashurambhau College,
Pune, India
anildusane@gmail.com
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