BIOLOGY-MDCAT-2024

ENZYMES

ENZYMES
• Introduction/characteristics of enzymes
• Mechanism of action of enzymes
• Factors effecting rate of enzyme action
• Enzyme inhibition
Learning Objectives
• Describe the distinguishing characteristics of enzymes
• Explain mechanism of action of enzymes
• Describe effects of factor on enzyme action (temperature, pH, concentration)
• Describe enzyme inhibitors

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 BIOLOGY-MDCAT-2024
ENZYMES

INTRODUCTION/CHARACTERISTICS OF ENZYMES
• Simply known as biological catalyst or biocatalyst
• Their name ends with
o –ase
▪ Lipase
▪ Sucrase
▪ Amylase
o –in
▪ Pepsin
▪ Trypsin
▪ Renin
• Mostly enzymes are of
o Globular proteins
▪ Amylase
• Some enzymes are of
o Polypeptides
▪ Pepsin
• Few enzymes are of
o RNA
▪ Ribozyme (found in ribosomes)
• It controls polypeptide elongation during protein synthesis such as peptidyl
transferase
• Enzyme are the most important group of proteins which are biologically active
• The term enzyme was coined by from Greek word ‘levened’ or ‘in yeast’
• First enzyme was discovered by Payen and Person from germinating barley seeds in 1833 and
named it as diastase (involved in the digestion of starch)
• The term enzyme was introduced by Wilhelm Kuhne in 1877
• Enzymes can be defined as the thermolabile biocatalyst protein in nature, specific in function and
coded by DNA

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ENZYMES
• Substrates are usually very smaller than enzymes
• They tremendously increase the efficiency of a biochemical reaction and are specific for each type
of reaction
• Without these enzymes the reaction would proceed at a very slow speed making life impossible
• Enzymes which catalyze chemical reaction again and again are called regulatory enzymes
• Enzymes are composed of hundreds of amino acids joined together and coiled upon themselves to
form a globular structure
• The catalytic activity is restricted to a small portion of the structure known as the active site, while
rest of the protein portion acts as framework
• The reactant called substrate is attached to the active site consisting of only a few amino acids,
while rest of the bulk of the amino acids maintains the globular structure of the enzyme
• Some enzymes consist solely of proteins
• Enzymes have relatively high molecular weight e.g.,
o Peroxidase (40 K Dalton)
o Catalase (250 K Dalton)
▪ One hydrogen atom has mass of one Dalton
• They can work in vivo (living cells) as well as in vitro (glassware)
• One enzyme may catalyze 100,000 substrates in on second (The unit is called as one turnover
number)
• Enzymes has three-dimensional structure with tertiary structure of protein
• Ribonuclease consists of 124 amino acids
• Many enzymes are simply dissolved in the cytoplasm
• Other enzymes are tightly bound to certain sub-cellular organelles
• They are produced by living cells for use in or near the site of their production
• The enzymes important in photosynthesis are found in the chloroplast
• The enzymes involved in cellular respiration are found in the mitochondria
• Some of the enzymes which are involved in the synthesis of proteins are integral part of ribosomes

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ENZYMES
• Most enzymes do not float about in a kind of cytoplasmic ‘soup’ but are attached to membrane
systems inside the cell in the specific and orderly arrangements. Mitochondria and chloroplasts
are good examples of this
• The minimum amount of energy required to start a reaction is called activation energy

• Some enzymes are potentially damaging if they are manufactured in their active form
• Pepsin is a powerful protein-digesting enzyme and is quite capable of destroying cell’s internal
structure and thus is produced in inactive pepsinogen form by the cell
• It is converted in its active form only in the digestive tract (stomach) where it is required to be
active
• Zymogen cells of stomach secretes pepsinogen
• Parietal cells of stomach secrete HCl

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ENZYMES

CO-FACTORS
• The non-protein part of an enzyme is known as a co-factor, which is essential for the proper
functioning of the enzymes
• The co-factor usually acts as ‘bridge’ between the enzyme and its substrate, often it contributes
directly to the chemical reactions which bring about catalysis
• Sometimes the co-factor provides a source of chemical energy, helping to drive reactions which
would otherwise be difficult or impossible
• Some enzymes are only composed of protein i.e., no co-factors are attached with them e.g., Lipase
• Co-factors are classified as

ACTIVATOR
• Some enzymes use metal ions as co-factors like Mg2+, Fe2+, Cu2+, Zn2+ etc.
• It is detachable co-factors
• It is an inorganic in nature
• Glucose + ATP (in the presence of hexokinase + Mg2+) forms Glucose-6-phosphate + ADP

PROSTHETIC GROUP
• It is covalently bonded
• It is inorganic or organic in nature
• e.g., haem, biotin etc.

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ENZYMES
• An iron containing porphyrin ring attached to some enzymes like cytochromes is the example of
prosthetic group

CO-ENZYME
• If it is loosely attached to the protein part it is known as coenzyme e.g., NAD, NADP, FAD, FMN
(Flavin mononucleotide), ATP, CoA etc.
• It is also detachable and are organic in nature
• Ethyl alcohol (in the presence of alcohol dehydrogenase + NAD+) forms acetaldehyde + NADH2
• It is closely related to vitamins, which represent the essential raw materials from which co-
enzymes are made
• Only small quantities of vitamins are needed because, like enzymes, co-enzymes can be used again
and again
• Lack of vitamin B produces beriberi
• A co-enzyme constitutes about 1 % portion of the entire enzyme molecule

Activators Prosthetic group Co-enzymes

Detachable Undetachable Detachable
Metallic ions Covalent or inorganic in nature Organic in nature
Ionic bond Strong covalent bond Weak linkages
Cannot be used again and again Cannot be used again and again Can be used again and again

APOENZYME AND HOLOENZYME

APOENZYME
• An enzyme with its co-enzyme or prosthetic group removed is designated as apoenzyme e.g.,
Pepsin, Trypsin, Proteozyme etc.
• Adding the correct concentrated co-enzyme to the apoenzymes will restore enzyme activity

Enzyme – cofactor = Apoenzyme

HOLOENZYME
• An activated enzyme consisting of polypeptide chain and a co-factor is known as holoenzyme e.g.,
DNA polymerase, RNA polymerase etc.
• Euler (1932) proposed that conjugated enzymes showing complete activity be called holoenzyme
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ENZYMES
Enzyme + cofactor = Holoenzyme

MECHANISM OF ACTION OF ENZYMES

• An enzyme is a three-dimensional globular protein that has specific chemical composition due to
its component amino acids and a specific shape
• Every enzyme by virtue of its specificity recognizes and reacts with a special chemical substance
called substrate
• Any enzyme, therefore, reacts only with its specific substrate and transforms it into product (s)
o Salivary amylase (it converts starch into maltose)
• Enzyme is released unaltered and thus can be used again and again
• The enzyme after catalysis detaches itself from the products unchanged
• Enzymes requires aqueous medium for its activity
• A site of an enzyme where substrate binds and is converted into product is known as active site of
an enzyme
• It consists of only a few amino acids (3-12 amino acids)
• It is a charge bearing site of an enzyme
• It is a polypeptide chain
• The active site of aldolase consists of glycine, histidine and alanine amino acids
• There are two regions of active site of an enzyme
o Binding site
▪ It recognizes the substrate
o Catalytic site

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ENZYMES
▪ It converts the substrate into product
• A site other than active site of an enzyme, where a substrate can bind but is not converted into
product
• It consists only of binding site
• It lacks catalytic site
• Enzymes with Allosteric sites are known as Allosteric enzymes

LOCK AND KEY MODEL

• Proposed by a German chemist Emil Fischer in 1894
• To visualize substrate and enzyme interaction
• Shape of enzyme is complementary to the shape of substrate
• According to this model,
o As one specific key can open a specific lock, in the same manner a specific enzyme can
transform only one substrate into product(s)
o The active site is a rigid structure
o There is no modification or flexibility in the active site before, during or after the enzyme
action and it is used only as a template
• This model was later improved by Paul Filder and D.D Woods
• This theory depends upon physical contact between substrate and enzyme molecule
• The specific action of enzyme with a single substrate can be explained using a lock and key
analogy
• The enzymes work on the mechanism called non-regulatory enzymes e.g., lipase, amylase etc.
• This model is exercised by a very small number of enzymes e.g., sucrase, maltase etc.
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ENZYMES
• When one enzyme can catalyze only one substrate and essentially no others it is called absolute
specificity e.g., urease
o Urea + Water (in the presence of urease) forms Ammonia + Carbon dioxide

INDUCE FIT MODEL

• Proposed by Daniel Koshland in 1958
• It is the modified form of lock and key model
• According to this model,
o When a substrate combines with an enzyme, it induces changes in the enzyme structure
o The change in structure enables the enzymes to perform its catalytic activity more
effectively
• Enzyme molecules are in an inactive form, in order to become activates it undergoes slight
configurational changes in the structure to accommodate the substrate.

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ENZYMES
• A suitable analogy would be that of hand and gloves
• The hand corresponds to the substrate and gloves as enzyme is shaped is inserted of the hand
• Enzymes which follow the induced-fit mechanism are called regulatory or allosteric enzymes e.g.,
hexokinase
• The example of carbonic anhydrate which can add oxygen to haemoglobin as well as can control
for formation of carbonic acid and bicarbonates in blood

FACTORS EFFECTING RATE OF ENZYME ACTION

• The functional specificity of every enzyme is the consequence of its specific chemistry and
configuration
• Any factor that can alter the chemistry and shape of an enzyme can affect its rate of catalysis
• Some of the important factors that can affect the rate of enzyme action are

ENZYME CONCENTRATION
• The rate of reaction depends directly on the amount of enzyme present at a specific time at
unlimited substrate concentration
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ENZYMES
• If the amount of enzyme is increased by two folds the reaction is doubled
• By increasing the enzyme molecules an increase in the number of active sites takes place
• More active sites will convert the substrate molecules into product(s), in the given period of time
• After a certain limiting concentration, the rate of reaction will no longer depend upon this increase
• Normally enzymes are present in cells in rather low concentrations
• If there is only one enzyme in the system it can convert hundreds of substrates into products but it
takes more time

SUBSTRATE CONCENTRATION
• At low concentration of substrate, the reaction rate is directly proportional to the substrate
available
• At higher concentrations the enzyme molecules become saturated with substrate, so there are few
free enzymes molecules, so added more substrate doesn’t make much difference
• If the enzyme concentration is kept constant and the amount of substrate is increased, a point is
reached when a further increase in the substrate does not increase the rate of reaction any more
• This is because at high substrate level all the active sites of the enzymes are occupied and further
increase in the substrate does not increase the reaction rate
• Substrate and enzyme concentration are directly proportional up to a certain maximum velocity
after which further increase in substrate concentration has no effect on the rate of reaction
• At certain concentration substrate become saturated then any further increase will have no effect
on the rate of reaction because at this point all the active sites of enzymes will be occupied,
maximum rate (V-max)
• The increase in substrate increases the velocity of the enzymatic reaction at first
• The reaction ultimately reaches a maximum velocity at equilibrium state
• The rise in velocity is decreased progressively with further increase in substrate
• The reaction does not increase by any further rise in substrate concentration, this happens
because all the active sites on enzyme molecules are occupied by the substate (saturation) and no
enzyme is left free to bind with additional molecules of the substrate

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ENZYMES

TEMPERATURE
• The rate of enzyme-controlled reaction may increase in temperature but up to a certain limit
• All enzymes can work at their maximum rate at a specific temperature called as optimum
temperature
o For enzymes of human body 37 OC is the optimum temperature
o For enzymes of plant body 25 OC is the optimum temperature
o For enzymes of bacteria living in hot springs 70 OC is the optimum temperature
o For enzymes of mammals 40 OC is the optimum temperature
o For enzymes of arctic snow flea -10 OC is the optimum temperature
o For enzymes of thermophilic bacteria 90 OC is the optimum temperature
• Heat provides activation energy and therefore, chemical reactions are accelerated at high
temperature
• Heat also supplies K.E to the reacting molecules, causing them to move rapidly
• Thus, the reactants move more quickly and chances of their collision with each other are increased
• However, further increase in heat energy also increases the vibrations of atoms which make up
the enzyme molecule
• If the vibrations become too violent, globular structure essential for enzyme activity is lost and the
enzyme is said to be denatured
• The rate doubles for each 10 OC rise in temperature
• The increase in rate with temperature can be quantified as a Q IO, which is the relative increase
for a 10 OC rise in temperature

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ENZYMES
• The rate is not zero at 0OC, so enzymes still work in the fridge (and food still goes off), but they
work slowly
• Enzymes can even work in ice, though the rate is extremely slow due to the very slow diffusion of
enzymes and substrate molecules through the ice lattice
• The temperature which causes denaturation of enzyme is called maximum temperature
• Such enzymes have been used in biological washing powders for high temperature washes
• If temperature is reduced to near or below freezing point, enzymes are inactivated not denatured
• The temperature where an inactive enzyme becomes active again is called minimum temperature

pH
• Every enzyme function most effectively over a narrow range of pH known as the optimum pH
• A slight change in pH can change the ionization of the amino acids at the active site
• Moreover, it may affect the ionization of the substrate
• Under this changed condition enzyme activity is either retarded or blocked completely
• Extreme changes in pH cause the bonds in the enzymes to break, resulting in the enzyme
denaturation
• Most of enzymes in our body work in the range of pH 6-8
• For most enzymes work about pH 7-8 (physiological pH of most cells), but few enzymes can work
at extreme pH, such as Protease enzyme in animal stomachs, which have an optimum pH 1.0
• Papain enzyme found in green papaya may work on both acidic or alkaline media

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ENZYMES
• Change in pH alters the ionic charge of acidic and basidic groups as a result ionic bonding is
disrupted

Enzymes Optimum pH
Protease 1.00
Pepsin 2.00
Invertase 4.50
Sucrase 4.50
Enterokinase 5.50
Maltase 6.10-6.80
Salivary amylase 6.80
Urease 7.00
Catalase 7.60
Trypsin 7.80-8.70
Chymotrypsin 7.00-8.00
Pancreatic lipase 9.00
Arginase 9.70

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ENZYMES

ENZYME INHIBITION
• An inhibitor is a chemical substance which can react (in place of substrate) with the enzyme but is
not transformed into product(s)
o It blocks the active site temporarily or permanently
• Examples of inhibitors are
o Poisons like
▪ Cyanide
o Antibiotics
o Anti-metabolites
o Some drugs
o Pesticides
o Research tools
o Toxins
o Products of enzymes
• Cyanides are powerful poisons of organisms because they can kill them by inhibiting cytochrome
oxidase essential for respiration. They block the action of these enzymes by combining with iron
which may be present in the prosthetic group
• Ions of heavy metals such as mercury, silver and copper combine with thiol (-SH) groups in the
enzyme breaking the disulphide bridges
• Penicillin blocks the active site of an enzyme unique to bacteria, when penicillin is taken, bacteria
die but human are unaffected
• Inhibitors can be divided into two types
1. Irreversible inhibitors
• They check the reaction rate by occupying the active sites or destroying the globular structure
• They occupy the active sites by forming covalent bonds or they may physically block the active
sites
• They often contain reactive functional groups e.g.,
o Aldehydes
o Alkanes
• The electrophilic groups make covalent bonds with amino acid side chains
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ENZYMES
• They may be natural or artificial e.g.,
o Poisons
o Venom of snakes
o Drugs etc.
2. Reversible inhibitors
• They form weak linkages or non-covalent bonds such as
o Hydrogen bonds
o Hydrophobic interactions
o Ionic bonds
• Their effect can be neutralized completely or partly by an increase in the concentration of the
substrate
• Feedback inhibition is an example of reversible non-competitive enzyme inhibition
• They are further divided into two major types
i. Non-competitive inhibitors
• They form enzyme inhibitor complex at a point other than the active site (Allosteric site)
• They alter the structure of the enzyme in such a way that even if genuine substrate binds the
active site, catalysis fails to take place
• Examples includes
o Poisons like cyanide,
o Heavy metal ions
o Some insecticides
ii. Competitive inhibitors
• Because of the structural similarity with the substrate, they may be selected by the binding sites,
but are not able to activate the catalytic site, thus product(s) are not formed
• With high concentration of inhibitors, the chances of inhibition are also high
• e.g., Malonic acid
• The Sulphonamides to an antibacterial-drugs which act as competitive inhibitors
• The importance of competitive inhibitors is
o It supports lock and key hypothesis

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ENZYMES
o It shows that substances which are similar to substrate are not acted by enzymes
o They are used as drugs in the control of bacterial pathogens

FEEDBACK MECHANISM
• In certain cases, enzymes act in a series of chemical reactions in a particular order to complete a
metabolic pathway such as respiration or photosynthesis.

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ENZYMES
• The successive enzymes containing these reactions are normally present together in a precise
order of reaction such that substrate molecules can be literally handed on from one enzyme to
another forming an enzyme-to-enzyme chain.
• In this way, the products from one step in pathway are transferred to the enzyme catalyzing the
next step.

• A precursor is a compound that participates in a chemical reaction that produces another

compound OR
• A chemical compound preceding in a metabolic pathway, such as a protein precursor

ENZYMES WITH THEIR ROLES

ENZYMES ROLE
Cellulase Digestion of cellulose
Pepsin Digestion of proteins in stomach
Sucrase Digestion of sucrose
Enterokinase It converts trypsinogen into trypsin
Salivary amylase/ Ptyalin In hydrolysis of starch in oral cavity
Catalase It catalyzes the decomposition of hydrogen peroxide to water and oxygen
Chymotrypsin Plays essential role in proteolysis or the breakdown of proteins and
polypeptides
Pancreatic lipase In hydrolysis and digestion of fat in duodenum
Arginase To detoxify ammonia in the urea cycle
Succinic dehydrogenase It catalyzes the oxidation of succinate into fumarate in the Krebs cycle
Acid phosphatase It catalyzes the hydrolysis of phosphate esters in an acidic environment

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ENZYMES
Peroxidase These enzymes are used to catalyze various oxidative reactions using hydrogen
peroxide and other substrates as electron donors
Glycolic acid oxidase It can serve to oxidize directly the glycolic acid produced in photosynthesis
Lysozyme It dissolves a portion of bacterial cell wall
Reverse transcriptase It converts single stranded RNA genome into double stranded viral DNA
ATP synthase It catalyzes the synthesis of ATP from ADP and phosphate
RUBISCO It catalyzes the first step of carbon fixation in the Calvin cycle of photosynthesis
Dehydrogenase It catalyzes the removal of hydrogen atom
Phosphofructokinase It catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-
bisphosphate
Pyruvate decarboxylase It catalyzes the decarboxylation of pyruvic acid to acetaldehyde
Pancreatic amylase/ Amylopsin It digests carbohydrates in small intestine
Trypsin/Proteolytic enzyme/proteinase It digests proteins in small intestine
Amino peptidase It catalyzes the cleavage of amino acids from the amino terminus of protein or
peptide substrates
Erypsin/Erepsin It digests peptones into amino acids
Maltase It catalyzes the hydrolysis of the disaccharide maltose to the simple sugar
glucose
Lactase It breaks lactose (sugar in milk and milk products)
Carbonic anhydrase It assists rapid interconversion of carbon dioxide and water into carbonic acid,
protons and bicarbonate ions
DNase DNA digesting enzyme
DNA polymerase I Plays a supporting role in DNA replication
DNA polymerase II It repairs the nucleotide base pairs to correct a sequence mistake during DNA
replication
DNA polymerase III It catalyzes replication of DNA strands
Primase It constructs RNA primer
DNA ligase It attaches the fragments to the lagging strand
DNA helicase It unwinds the two strands of DNA
RNA polymerase I It synthesizes rRNA
RNA polymerase II It synthesizes mRNA
RNA polymerase III It synthesizes tRNA
Nuclease It cleaves the phosphodiester bonds of nucleic acids
Phosphatase It removes a phosphate group from a protein
Aminoacyl-tRNA synthetase It attaches tRNA to a specific amino acid

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ENZYMES
Phenylalanine hydroxylase Is responsible for the conversion of phenylalanine to another amino acid,
tyrosine
Restriction endonucleases It is used to cut the chromosome on the flanking sites of the gene
Restriction enzyme To restrict the growth of virus/To cut the DNA at very specific sites
characterized by palindromic sequence
DNA ligase The enzyme that links together Okazaki fragments in DNA replication of the
lagging strand. It also links other broken areas of the DNA backbones. It joins
wo DNA molecules together end to end through phosphodiester bonds
Taq polymerase It automates the repetitive step of amplifying specific DNA sequence in PCR
Luciferase It produces light when they oxidize their substrate
Ligase Enzyme that joins together two molecules in an energy dependent process
RNA polymerase An enzyme that catalyzes the assembly of an mRNA molecule, sequence of
which is complementary to a DNA molecule used as a template

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