ISLAMIC UNIVERSITY

COURSE NO: 2205

COURSE NAME: ENZYMOLOGY
CHAPTER 4

REGULATORY & CATALYSIS

STRATEGIES

WRITTEN BY
ORIGIN
BTGE, IU
 Regulatory enzymes
Regulatory enzymes: A regulatory enzyme is an enzyme in a biochemical pathway, which
through its responses to the presence of certain other biomolecules, regulates the pathway
activity. This is usually done for pathways whose products may be needed in different
amount at different times such as hormone production. Regulatory enzymes exist at high
concentrations (low Vmax). So, their activity can be increased or decreased with changes
in substrate concentration. Example. Glycogen phosphorylase, Hexokinases,
phosphofructokinase, glutamine synthetase, aspartic transcarboxylase (ATCase)
➢ In a multi-step enzymatic process, there will be one enzyme which will be responsible
for the overall rate of that process. This critical rate limiting enzyme is called the
regulatory enzyme.
➢ It shows enhanced or decreased catalytic activities in response to other molecules
(signals) in the cells.
➢ In cellular metabolic activities, many enzymes work together in a sequence to carry out
the given metabolic process.
➢ Example, glycolysis pathway includes ten sequential steps each catalyzed by specific
enzymes.

Have extraordinary catalytic power.

➢ Cause thousand-fold increase in chemical reactions.
➢ Can convert millions of substrate molecules in to products in a fraction of time.
➢ It is essential to control the activity of enzymes in order to regulate the metabolic
activities in the cell.
➢ Permit changing needs of the cell to meet its energy.
➢ If the product is available in excess enzyme regulation could then convert to other
reactions.
➢ If product is available in demand, it could activate pathways to produce more of the
biomolecule that is needed.
➢ It uses ATP for its action.

Enzyme Regulation
❖ Control of the rate of a reaction catalyzed by an enzyme by some effector (eg, inhibitors
or activators) or by alteration of some condition (e.g., pH or ionic strength).
❖ If a product is available in excess, enzyme regulation could then divert the resources to
other needy reactions.

❖ When an effector binds to an enzyme, it is called cooperative binding.

 Enzyme Regulation Methods
1. Allosteric (Allosteric regulation of-enzymes).
2. Reversible covalent modification of enzymes.
3. Proteolytic activation of enzyme.
4. Feedback regulation.
5. Regulation by isoenzymes (isozymes).

Allosteric enzymes (Activator of Inhibitor)

Definition: Allosteric means "other site" or "other structure". Allosteric enzymes are
enzymes that have an additional binding site for effector/modulator molecules other than
the active site. The binding brings about conformational changes, thereby changing its
catalytic properties. The effector molecule can be an inhibitor or activator.

Properties of Allosteric Enzyme:

1. The biological catalyst, which increases or decreases the rate of the reaction.
2. Have an additional site, other than the active site or substrate binding site. The substrate-
binding site is known as C subunit or Catalytic subunit and effector/modulator binding
site is known as R-subunit or regulatory subunit.
3. Can be more than one allosteric site present in an enzyme molecule.
4. Have an ability to respond to multiple conditions, that influence the biological reactions.
5. The binding molecule is called an effector or modulator, it can be inhibitor as well as
activator.
6. The binding of the effector molecule changes the conformation of the enzyme.
7. Activator increases the activity of an enzyme, whereas inhibitor decreases the activity
after binding.
8. The velocity vs substrate concentration graph of allosteric enzymes is S. curve
(Sigmoidal) as compared to the usual hyperbolic curve.
9. Allosteric effectors bind non-covalently to the enzyme they regulate.
10.Effectors need not have any structural resemblance to substrate structure.
11.Do not follow the Michalis-Menten kinetics.
Types of Allosteric Enzyme:
On the basis of substrate and effector molecules
1. Homotropic Regulation: The substrate molecules act as an effector also. They are
mainly enzyme activation and known as cooperativity.
Example: Binding of O, to hemoglobin.
2. Heterotropic Regulation: The substrate and effector are different.
Example: Binding of CO₂ to hemoglobin.

On the basis of action performed by the regulator

1. Allosteric Inhibition: Inhibitors bind with protein due to which all active sites of
protein undergo conformational changes due to which activity of enzyme decreases.
2. Allosteric Activation: Activator binds with protein which increases the function of
active sites leads to increase in enzymatic activity.
Mechanism of Allosteric Regulation:
Two models proposed for the mechanism of allosteric regulation:
1. Simple Sequential Model:
It was given by Koshland. In this model, the binding of substrate induces a change in the
conformation of the enzyme from T (tensed) to R (relaxed) The substrate binds according
to the induced fit theory. A conformational change in one unit stimulates similar changes in
other subunits. This explains the cooperative binding, same way inhibitors and activators
bind, the T form is favoured, when the inhibitor binds and R form is favoured, when the
activator binds. The binding at one subunit affects the conformation of other subunits. The
sequential model explains the negative cooperativity in enzymes, eg tyrosyl tRNA
synthetase, where the binding of substrate inhibits the binding of another substrate.

2. Concerted or Symmetry Model:

This model was proposed by Monad, According to this model, there is a simultaneous
change in all the subunits of an enzyme. All the subunits are either present in R form (active
form) or T form (inactive form), having less
affinity to substrate. An inhibitor shifts the
equilibrium of TR, towards T, and activator
shifts the equilibrium towards R form and
favours the binding. It explains the
cooperative regulation of activators as well as
inhibitors (S=substrate, A=agonist, l-
inhibitor).
Examples of Allosteric Enzyme:
Aspartate Transcarbamoylase (ATCase)
➢ ATCase catalyzes the biosynthesis of pyrimidine.
➢ Cytidine triphosphate (CTP) is the end product and also inhibits the reaction. It is
known as feedback regulation.
➢ High concentration of ATP can overcome inhibition by CTP.
➢ This ensures the synthesis of pyrimidine nucleotide when a high concentration of
purine nucleotide is present.
Glucokinase
➢ It plays an important role in glucose homeostasis. It converts glucose to glucose-6-
phosphate and enhances glycogen synthesis in the liver. It also senses the
concentration of glucose for the release of insulin from pancreatic beta cells.
➢ It has low affinity for glucose, so it acts when more concentration of glucose is present
in the liver, which should be converted to Glycogen.
➢ The activity of glucokinase is regulated by glucokinase regulatory protein.
Acetyl-CoA Carboxylase
➢ It regulates the process of lipogenesis.
➢ It is activated by citrate and inhibited by a long chain acyl-CoA molecule such as
palmitoyl-CoA, which is an example of negative feedback inhibition by product.
➢ It is also regulated by phosphorylation/ dephosphorylation controlled by hormones
such as glucagon and epinephrine.
Phosphofructokinase (PFK)
➢ It is the key monitoring enzyme in glycolysis and the cellular production of energy
from the disintegration of carbohydrate molecules.
➢ Glycolysis results in the generation of pyruvate and high-energy molecules,
adenosine triphosphate (ATP).
Isocitrate dehydrogenase (IDH)
➢ It catalyzes the primary regulatory part of the citric acid cycle.
➢ Also known as the Krebs cycle or the tricarboxylic acid (TCA) cyde, it is the
regulatory pathway in aerobic metabolism, which gains energy from the
embarrassment of acetyl CoA and provides building units for the biosynthesis of
amino acids, heme, and nucleic acids.
➢ Human IDH has two sets of heterodimers, one acts as a catalytic subunit and the other
as the regulatory subunit.
 Feedback inhibition
Definition: It is defined as (also called end product Inhibition) the process in which the
end product of a reaction Inhibits or controls the action of the enzyme that helped produce
it. In other words, the end products formed in the reaction actually get enzymes to slow
down or stop making new products altogether.

Feedback inhibition work:

End Feedback inhibition is a cellular control mechanism in which an enzyme’s activity is
inhibited by the enzyme’s end product. This mechanism allows cells to regulate how much
of an enzyme’s end product is produced. Most biochemical processes are complex and
multi-step, requiring multiple enzymes to get from the starting substrate to the desired end
product. Typically, feedback inhibition acts on the first enzyme unique to a given pathway.
For example, in the case of amino acid production, an amino acid may act as an inhibitor
for the first enzyme in the pathway whose purpose is making more of that amino acid.
Binding of a regulatory messenger – in this case, the end product of the biochemical
pathway – to the allosteric site changes the shape of the whole enzyme. In feedback
inhibition, binding of the end product to the allosteric site slows down or stops the enzyme’s
activity so that little or no new end product is produced. When levels of the end product
drop, the enzyme will encounter fewer particles of the end product and its activity will
increase again. Feedback inhibition prevents waste that occurs when more of a product is
made than the cell needs. It can also prevent harm when having too much of the pathway’s
end product may actually be harmful to the organism.
Feedback Inhibition Example
1. Cholesterol production:
Feedback inhibition controls cholesterol production. It is important to facilitate signaling
between cells and maintain the integrity of cell membranes. However, too much of
cholesterol can be pretty dangerous, can lead to terrible consequences. Therefore, it
becomes important for the body to reduce the formation of cholesterol when it reaches
dangerous levels. That's where feedback inhibition helps. If a lot of cholesterol is present
in the bloodstream, on new cholesterol producing enzyme is made, which eventually
leads to a gradual decline in the cholesterol levels in the body.
2. Amino Acid Synthesis:
Amino acids are "building blocks" of protein that the human body needs. However,
various amino acids may be required by the cell at various periods. This means, similar
to transforming glucose to ATP, cells must figure out how to make the most efficient use
of their basic materials to produce precisely what they require at any given time. They
utilize feedback regulation, just like ATP, to ensure that they only create the amino acids
required at any particular time.
3. Regulation of ATP production:
ATP or Adenosine Triphosphate is formed from glucose due to a series of enzymatic
reactions within our cells. If you have too much ATP produced, it could lead to glucose
depletion in your body and energy loss. Therefore, it's important to regulate the amount
of glucose that is broken down to produce ATP. ATP (10 steps of glycolysis) binds to a
particular enzyme (phosphofructokinase, 3 steps glycolysis), which stops from further
breakdown of glucose stored in the body.

Function:
1. Waste: Without It, energy or raw materials that could be used for important cellular
functions might be wasted on unnecessary ones.
2. Prevents depletion: Without It, raw materials and energy might be depleted by
biochemical processes that don't stop, even when their end product is not needed.
Example-the production of ATP from glucose. The enzymes that produce ATP from
glucose are subject to feedback inhibition by ATP. This saves glucose by preventing
its unnecessary breakdown when the cell has plenty of ATP.
3. Prevents dangerous build-up: The end products of some biochemical pathways can
be dangerous in high concentrations. Cholesterol is an excellent example of our body
can make that is good in small quantities but dangerous in large quantities.
4. Maintain homeostasis: An essential function of life is ability to maintain constant
internal circumstances in the face of changing environmental circumstances. Some
chemical messengers that are involved in maintaining homeostasis are regulated
through feedback regulation.
Mechanism of Aspartate Transcarbamoylase/ How are allosteric enzymes controlled?
ATCase catalyzes the first step in a series of reactions in which the end product is cytidine
triphosphate (CTP), a nucleoside triphosphate needed to make RNA and DNA. The
pathways that produce nucleotides are energetically costly and involve many steps. The
reaction catalyzed by aspartate transcarbamoylase is a good example of how such a pathway
is controlled to avoid overproduction of such compounds. For DNA and RNA synthesis, the
levels of several nucleotide triphosphates are controlled. CTP is an inhibitor of ATCase, the
enzyme that catalyzes the first reaction in the pathway. This behaviour is an example of
feedback inhibition, in which the end product of the sequence of reactions inhibits the first
reaction in the series.
 Covalent modification
Definition: Covalent modifications are enzyme-catalyzed alterations of synthesized
proteins and include the addition or removal of chemical groups. Modifications can target
a single type of amino acid or multiple amino acids and will change the chemical properties
of the site.
Enzymes can be regulated by transfer of a molecule or atom from a donor to an amino acid
side chain that serves as the acceptor of the transferred molecule. Another way of regulating
an enzyme is by altering the amino acid sequence itself by proteolytic cleavage.
Most common modifying groups include:
1. Phosphorylation (most common).
2. Adenylylation
3. Acetylation
4. Miristoylation
5. Ubiquitination (important for protein degradation)
6. ADP-ribosylation
7. Methylation
 Phosphorylation
Addition of phosphate group to Tyr, Ser, Thr and His residue of protein.
➢ It is a regulatory covalent modification.
➢ Addition of phosphoryl group to specific amino acid residues (Tyr, Ser, Thr, His) of
A protein.
➢ Catalyzed by the enzyme protein kinase.
➢ ATP or GTP act as phosphate group donor.
➢ Energy derived from the cleavage of phosphate group donor is also utilized.
➢ Some proteins have phosphorylated residue, other have several sites for
phosphorylation.
➢ Only moderately polar group is changed.
➢ One third to one half of all proteins in eukaryotic cells are phosphorylated.

Adenylation
Adenylation is an elegant biological process used to chemically activate carboxylate
substrates by condensing them with ATP to liberate pyrophosphate.
Methionine is an essential sulfur-containing gluconeogenic amino acid that is converted to
S-adenosylmethionine (SAMe. abbreviated also as Ado-Met or SAM) by the enzyme
methionine adenosyl transferase (MAT) using ATP as co-substrate.
 Acetylation
Addition of acetyl group to Lys residue of protein.
➢ Lysine acetylation, which is catalyzed by KATS, involves transfer of an acetyl from
Ac-CoA to the e-amino side chain of lysine or occurs nonenzymatically.
➢ Deacetylation of lysine residues is catalyzed by Zn2+ -dependent HDACs or by NAD-
dependent SIRTS. HDACs=Histones Deacetylate, KATS = Lysine acetyltransferases,
SIRTS =NAD dependent HDACs, also known as sirtuins.

ADP-ribosylation
It is a type of protein posttranslational modification initiated by a group of enzymes named
poly (ADP ribose) (PAR) polymerases (PARPS)Posttranslational modification of proteins
by reversible ADP ribosylation. Mono- and poly ADP-ribosyl transferases (ARTS and
PARPS) catalyze the transfer of an ADP-ribose moiety from B NAD to a target amino acid
side chain or previously linked ADP ribose on a protein, releasing nicotinamide in the
process. The reverse reaction is catalyzed by mono- and poly-(ADP-ribosyl)
glycohydrolases (ARHS and PARGS), that cleave the a glycosidic bond between C1' and
the protein side chain or the preceding ADP- ribose.
 Myristoylation
It is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid,
myristic acid, to the N-terminal glycine of a subset of proteins.
 Ubiquitination
It involves the attachment of ubiquitin to lysine residues on substrate proteins or itself by
E3 ligases, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin
attachment to different lysine residues can generate diverse substrate-ubiquitin structures,
targeting proteins to different fates.

Methylation
A chemical reaction in the body in which a small molecule called a methyl group gets added
to DNA, proteins, or other molecules. The addition of methyl groups can affect how some
molecules act in the body. For example, methylation of the DNA sequence of a gene may
turn the gene off so it does not make a protein. Changes in the methylation patterns of genes
or proteins can affect a person's risk of developing a disease, such as cancer.
The mechanism of DNA methylation. DNA methylation is exerted by DNA
methyltransferases (DNMTs). DNMTS at the 5-position of cytosine residues in CpG
dinucleotides transfers methyl groups from SAM (S-adenosylmethionine) to SAH (S
adenosylhomocysteine); thus, 5-methylcytosine is formed.
Protease Enzyme Name Functions

Trypsin Found in pancreatic juice and breaks proteins and

peptones and proteoses to dipeptides.

Chymotrypsin Found in pancreatic juice and breaks proteins and

peptones and proteoses to dipeptides.

Carboxypeptidase Found in pancreatic juice and breaks proteins and

peptones and proteoses to dipeptides.

Elastase Present in pancreatic juice and digests elastin.

Nuclease (ribonuclease and Present in pancreatic juice. They split nucleic acid to
deoxyribonuclease) nucleotides.

Collagenase It digests collagen.

Dipeptidase Found in intestinal secretion. Breaks dipeptides to amino
acids.

Pepsin Present in stomach and converts proteins to smaller

peptides-proteoses and peptones.

Rennin Secreted by chief cells of the stomach and curdles milk

protein.

Thrombin Involved in blood coagulation.

Plasmin Involved in blood coagulation.
Renin Secreted by juxtaglomerular cells of the kidney and
converts angiotensinogen to angiotensin.

Hyaluronidase Present in the acrosome of sperms and helps in penetration

of sperm into the ovum during fertilization.

Insulinase Present in the kidney and liver. It degrades insulin.

Chymases, tryptases They are present in mast cells and involved in allergic
reactions and inflammation.

Cathepsin Neurolysin Present in immune cells.

 Proteolytic enzymes or Protease
It is an enzyme that catalyzes the hydrolysis of peptide bonds present in proteins. In most
of the living organisms, protease enzymes are essential for digestion and absorption of
proteins. It is found in all living organisms, eg C bacteria, algae, plants and animals and in
some of the viruses too. They are involved in the catabolism and digestion of proteins and
also in cell signaling.
Types:
Exopeptidase: It catalyzes the deavage on terminal peptide bond. e.g. aminopeptidases,
carboxypeptidases, etc.
Endopeptidase: It facilitates the deavage of internal peptide bonds of proteins. e.g. pepsin,
chymotrypsin, elastase, T etc.

Functions:
➢ They are proteolytic, they help in digestion and catabolism of proteins. They catalyze
the hydrolysis of peptide bonds and convert them to amino acids, which is then absorbed
and utilized by cells.
➢ They are required for the blood coagulation process.
➢ Protease enzymes Involved in the cell division, growth,apoptosis and migration
➢ Protein recycling transport across membranes.
➢ They are involved in the activation of precursor proteins and zymogens.
➢ Proteases provide immune support and regulate the process of tumour growth,
metastasis, inflammation, etc. They may help in wound healing and muscle soreness.

Protease work:
➢ It catalyzes the hydrolysis of peptide bonds. Catalysis facilitates the nucleophilic attack
of an activated water molecule on the peptide bond.
➢ Serine, cysteine and threonine proteases function by forming an acyl-enzyme
Intermediate, which then gets hydrolyzed by water to get the product and enzyme is set
free.
➢ It ranges from general to specific, e.g. digestive protease enzyme, trypsin can deave
many proteins into smaller fragments, whereas enzymes like thrombin, which takes part
in blood clotting are highly specific.
➢ Many protease enzymes are present in an Inactive form. Being proteins themselves, these
precursors get converted to an active form by another protease enzymes. It helps in the
regulation and control of the activity Eg. trypsinogen, chymotrypsinogen,
procarboxypeptidase, proelastase etc.
 Isozymes or Isoenzymes
Definition: Multiple forms of enzymes that differ in amino acid sequence but catalyze the
same chemical reaction. It was described by R. L. Hunter and Clement Markert in the year
1957. a-amylase, glucokinase, lactate dehydrogenases all are examples of isozymes.
Characteristics of Isozymes:
➢ Enzyme variants are the product of different genes and thus represent different loc.
➢ They are the result of gene duplication but can also arise from polyploidization or nucleic
acid hybridization.
➢ They have different kinetic parameters (eg. different K values) or are regulated
differently.
➢ They catalyze the same reaction, but they can be distinguished by physical methods such
as electrophoresis or by immunological methods.
➢ The difference between some isozymes is due to differences in the quaternary structure
of the enzymes, e.g., lactate dehydrogenase exists in five isozymic forms.
➢ The isozymic forms of lactate dehydrogenase are tetramers, each is made up from two
types of units H and M. The molecular weight of active lactate dehydrogenase is
1,30,000. Only the tetrameric molecule possesses catalytic activity.
Isozymes Work:
Isozymes are variations of an enzyme that are produced by Isozymes can be defined as
distinct versions of an enzyme that are encoded by various genes. Isozymes' structural
differences from their amino acid sequences are marginal. As a result, they come in a variety
of sizes and forms. However, they both catalyze the same biological process.
However, they are able to function in a number of circumstances, at various points in our
bodies, at various phases, or in varied cellular situations.
Types:
1. CPK (creatine phospho kinase)
2. LDH (Lactate Dehydogenase)
3. Troponin
4. ALP (Alkaline phosphatase)
5. Aldolase
6. Amylase
CPK
Also known as creatine kinase (CK), is an enzyme expressed by various tissues and cell
types. CK catalyses the conversion of creatine and uses ATP to create phosphocreatine (PC)
and ADP. This CK enzyme reaction is reversible and thus ATP can be generated from PC
and ADP.
Why is the Test Performed? /What happens when CPK levels are high?
When the total CPK level is very high 125 ug/L (Normal values: 10 to 120 ug/L), it means
there has been injury or stress to muscle tissue, the heart, or the brain. Muscle tissue injury
is most likely. When a muscle is damaged, CPK leaks into the bloodstream. Finding which
specific form of CPK is high helps determine which tissue has been damaged. High CPK
levels may be seen in people who have Brain injury or stroke, Convulsions, Delirium
tremens, Dermatomyositis or polymyositis, Electric shock, Heart attack, Inflammation of
the heart muscle, Lung tissue death (pulmonary infarction). Muscular dystrophies,
Myopathy, Rhabdomyolysis, Trauma.

Different types of CPK & their side

1. CPK-1: (also called CPK-BB) is found mostly in ns the brain and lungs.
Side effects: Stroke or bleeding in the brain, a seizure, brain cancer, a pulmonary
infarction, or the death of lung tissue.
2. CPK-2: (also called CPK-MB) is found mostly in the heart.
Side effects: injury to your heart, inflammation of your heart muscle, an
electrical injury, a heart attack
3. CPK-3: called CPK-MM) is found mostly In skeletal muscle.
Side effects: Damaged from a crush injury, immobile for an extended period,
damaged by illegal drug use, inflamed.
 LDH
It is an enzyme found in nearly all living cells. LDH catalyzes the conversion of lactate to
pyruvate and back, as it converts NAD to NADH, and back A dehydrogenase is an enzyme
that transfers a hydride from one molecule to another.

LDH produce ATP

➢ Mitochondria is the powerhouse of cells that are generally involved in the production of
energy currency which is ATP.
➢ ATP apart from energy production also synthesize the macromolecules that are required
by the cell for their survival.
➢ ATP can be used to switch to control chemical reactions and send messages.

Types of LDH

Isoenzyme Composition Present

names
LDH1 HHHH (H) Heart RBC and brain

LDH 2 HHHM (H3M1) Myocardium, RBC

LDH3 HHMM (H2M2) Kidney, skeletal muscle, lung

LDH4 HMMM White blood cells, kidney, pancreas cells, and

(H1M3) lymph nodes
LDHS MMMM (M4) Liver and muscles of skeleton.
LDH function
➢ It is an enzyme that is present in almost all living organisms.
➢ Its main function is to convert the end product of glycolysis which is pyruvic add into
lactic acid.
➢ This conversion by lactate dehydrogenase takes place in the absence of oxygen.
➢ During this conversion process, hydrogen is released from NADH, forming NAD'
molecule, also two molecules of ATP are generated during the conversion of glucose to
pyruvate.
➢ It functions in maintaining homeostasis when anaerobic conditions arise in the body.

Important of LDH
➢ LDH is a particular kind of protein that is an enzyme. • LDH is crucial in the production
of the body's energy. Nearly every tissue in the body, including those in the blood, heart,
kidneys, brain, and lungs, contains it.
➢ LDH is released into the bloodstream or other bodily fluids when these tissues are
injured. As it catalyzes the conversion of NAD+ to NADH and back, LDH also
transforms lactate to pyruvate and back.
➢ A dehydrogenase is an enzyme that transfers a hydride from one molecule to another.
➢ The crucial enzyme lactate dehydrogenase (LDH) aids in the process of cellular
respiration, which is how our body converts the glucose (sugar) from the food we eat
into energy for our cells.
➢ Proteins are called enzymes to aid in accelerating our body's chemical reactions, or
metabolism.
➢ A crucial enzyme in anaerobic respiration is LDH.

Lysozymes
It is an enzyme present in both animal and human lacrimal gland secretions (or tears), gastric
secretions, nasal mucus, and egg white. It is discovered in 1921 by Sir Alexander Fleming.
The catalyze the breakdown of certain carbohydrates (breaks glycosidic linkages in
peptidoglycans) that are found in the cell walls of certain bacteria (for example, cocci). As
a result, In the case of lacrimal fluid, it protects the cornea of the eye from Infection
Lysozyme is most effective against the bacteria of Gram positive.
Mechanism of action of lysozyme:
The function of lysozyme is to hydrolyze the B(1-4) glycosidic bond between residues of
N-acetylmuramic acid (NAM) and N acetylglucosamine (NAG) in certain polysaccharides.
➢ Chitin, which is a polymer of NAG linked by B(1-4) glycosidic bonds. Chitin is found
in crustacean shell tissue.
➢ The polysaccharide component of the cell walls of certain bacteria; the principal function
of lysozyme is as an antibacterial agent. This polysaccharide is composed of alternating
residues of NAG and NAM. Only the glycosidic bonds between C-1 of NAM and C-4
of NAG are hydrolyzed.

Functions:
➢ Immune system: It destroys viruses and bacteria that attack the cell.
➢ Demolition squads: It is destroys worn out cellular organelles and organic debris.
(Autolysis)
➢ Suicide Bags: When a cell becomes old or is damaged, lysosomes burst and
enzymes digest their own cells.
➢ During starvation, lysosomes digest stored proteins, fats.

Properties:
➢ It has the characteristics of a ferment. The rapidity of its action increases up to 60°C,
but at temperatures over 65°C. it is destroyed more or less rapidly.
➢ It acts best in a neutral medium.
➢ Peptic or tryptic digestion does not destroy Lysozyme.
➢ Stability-When kept dry. It can be preserved for a long time. It was noted that
commercial dried egg albumen was very rich in Lysozyme.
Lysosomes known as Suicidal Bags:
As stated before, lysosomes work as the waste discarding structures of the cell by processing
undesirable materials and degrading them, both from the exterior of the cell and waste
constituents inside the cell. But sometimes, the digestive enzymes may end up damaging
the lysosomes themselves, and this can cause the cell to die. This is termed as autolysis,
where "auto" means "self" and "lysis" means "the disintegration of the cell by the
destruction of its cell membrane". Hence, lysosomes are known as "Suicidal Bags" of the
cell.

Chymotrypsin
It is a digestive enzyme that breaks down proteins (Le, it is a proteolytic enzyme; referred
to as a protease). It is naturally produced by the pancreas in the human body. However, it
can also be taken as an enzyme supplement to improve health and digestion and aid in the
treatment of various diseases.

Function of Chymotrypsin:
➢ It is a digestive enzyme synthesized in the pancreas and plays an essential role in the
proteolysis or degradation of proteins and polypeptidase.
➢ As the component of pancreatic juice, It deaves peptide amide bonds to help digest
the protein in the duodenum.
➢ The function of the chymotrypsin is to cleave the dietary protein and enters the
digestive tract.
➢ It is secreted from the pancreas and activated once it reaches the small intestine.

Chymotrypsin as medicine:
It is taken orally, inhaled, injected or applied to skin. Used to treat:
→ Ulcers
→ Shingles and acne
→ Surgical or traumatic injuries
→ Necrotic tissue
→ Help loosen phlegm in asthma, bronchitis, lung diseases, and sinus infections.
→ Wound
→ Fracture and burn treatments
→ Arthritis and such other autoimmune diseases as lupus, scleroderma, and multiple
sclerosis.
→ Pelvic Inflammatory diseases shingles
Mechanism of action of Chymotrypsin:
➢ The substrate enters the chymotrypsin active site and is held by hydrophobic interactions.
➢ The enzyme executes a nucleophilic attack on the carbonyl carbon of the substrate's
amino acid to form the first intermediate. A nucleophilic attack occurs when one
molecule donates or shares its electrons with another molecule.
➢ A carbonyl carbon is a carbon that is double bound to oxygen and single bound to another
carbon. An aromatic amino acid has a ring within its molecular structure. Phenylalanine,
tyrosine, and tryptophan are aromatic amino acids Aromatic amino acids are naturally
hydrophobic, therefore, they are attracted to the hydrophobic region of the chymotrypsin
active site.
➢ The first Intermediate is converted into a second Intermediate called the acyl-enzyme
intermediate. An acyl group is a specific kind of carbonyl group Chymotrypsin cleaves
a portion of the substrate, and it is free leave the active site. Specifically, it cleaves only
the hydrophobic portions of protein. Meanwhile, a second portion of the substrate
remains in the active site.
➢ Water enters the active site and performs a nudeophilic attack on the acyl-enzyme
intermediate. This action sparks a series of conformational changes until the enzyme
releases the remaining portion of the substrate.
 RNase
RNases (or ribonucleases) are class of hydrolytic enzymes that catalyzes both the in vivo
and in vitro degradation of ribonucleic acid (RNA) molecules Into smaller components. The
nuclease operates at the level of transcription and translation and breaks down the RNA by
cleaving the phosphorus-oxygen bonds.

RNase enzymes are categorized into two groups:

1. Exoribonucleases: It is an exonuclease ribonuclease that degrades RNA by removing
terminal nucleotides from either the 5' end or the 3' end of the RNA molecule. It has six
families of nucleases, including members such as RNase R RNase T, and RNase D.
2. Endoribonucleases: It cleaves RNA molecules internally. It can cleave either single-
stranded or double-stranded RNA depending on the enzyme. It has several forms that
structurally consist of either single proteins and or a complex of proteins with RNA.

Functions of RNase
➢ RNase 3 is actively involved in the regulation of transcription and mRNA lifetime.
➢ RNase L is Interferon-induced RNase that destroys all RNA within a cell.
➢ RNase T is the major contributor to the 3-to 5' maturation of many stable RNAs.
➢ RNase R can degrade RNA with secondary structures without any help from accessory
factors.
➢ RNase H cleaves 3'- O-P RNA bonds in an RNA/DNA hybrid duplex to form 3'-
hydroxyl and 5'- phosphate terminated products.

RNase Work:
➢ RNase A is a small protein having 124 amino acid residues with no attached
carbohydrate. Its structure involves 19 out of 20 amino acids, leaving tryptophan. It
cleaves the phosphodiester linkage between the 5-ribose of a nucleotide and the
phosphate group attached to the 3 ribose of an adjacent pyrimidine nucleotide. The
resulting 2 3-cyclic phosphate is hydrolyzed to the corresponding 3 nucleoside
phosphate.
➢ The His12 of ribonuclease acts as a base after accepting the 2- OH proton of the sessile
ribonucleic sugar ring.
➢ The reaction promotes a nucleophilic attack by the 2-0 on the more positively charged
phosphorus (P) atom, thus creating 2- 3'-cydic ribonucleotide phosphate an intermediate.
➢ The intermediate formation is also facilitated by the Imidazole of His119. It acts as a
general acid by donating its proton to the oxygen atom in the susceptible P-0-R' bond.
Then, the acid-base activities of the side-chains of both histidine's are reversed.
➢ His 12 acts as a general acid and donates its newly acquired proton to the 2-3'-cyclic
ribonucleotide phosphate intermediate. Conversely, His119 acts as a general base and
accepts a proton from water to promote hydroxyl attacks on the same 2-3'-cyclic
intermediate.