ENZYME

“Enzymes can be defined as biological polymers that catalyze biochemical reactions.”

The vast majority of enzymes are proteins with catalytic capabilities that are essential for maintaining various life
processes. Metabolic processes and other chemical reactions in the cell are carried out by a set of enzymes that are
necessary to sustain life.

The initial stage of metabolic process depends upon the enzymes, which react with a molecule and is called the
substrate. Enzymes convert the substrates into other distinct molecules and are called the products. The regulation of
enzymes has been a key element in clinical diagnosis because of their role in maintaining life processes.

The macromolecular component of all enzymes consists of protein, except in the class of RNA catalysts called
ribozymes. The word ribozyme is derived from the ribonucleic acid enzyme. Many ribozymes are molecules of
ribonucleic acid which catalyze reactions in one of their own bonds or among other RNAs.

Enzymes exist in all fluids and tissues of the body. Intracellular enzymes catalyze all the reactions that occur in
metabolic pathways. The enzymes in plasma membrane regulate catalysis in the cells in response to cellular signals and
enzymes in the circulatory system regulate clotting of blood. Almost all the significant life processes are based on the
enzyme functions.
 ENZYME STRUCTURE

Enzyme Structure: Enzymes are a linear chain of amino acids that generate the three-dimensional structure. The
sequence of amino acids enumerates the structure which in turn identifies the catalytic activity of the enzyme. The
structure of the enzyme denatures when heated, leading to loss of enzyme activity, which is typically connected to the
temperature. Enzymes are larger than their substrates and their size vary, which range from sixty-two amino acid
residues to an average of two thousand five hundred residues present within fatty acid synthase. Only a small section of
the structure is involved in catalysis and are situated next to binding sites. The catalytic site and binding site together
constitute the enzyme’s active site. A small number of ribozymes exists which serves as an RNA-based biological
catalyst. It reacts in complex with proteins.
 Classification of Enzyme
According to the International Union of Biochemists (I U B), enzymes are divided into six functional classes and
are classified based on the type of reaction in which they are used to catalyze. The 6 types of enzymes are

Following are the enzymes classifications in detail:

Types Biochemical Property

The enzyme Oxidoreductase catalyzes the oxidation reaction where the electrons tend to travel
1. Oxidoreductases
from one form of a molecule to the other.

The Transferases enzymes help in the transportation of the functional group among acceptors
1. Transferases
and donors molecules.

Hydrolases are hydrolytic enzymes, which catalyze the hydrolysis reaction by adding water to
1. Hydrolases
cleave the bond and hydrolyze it.

Adds water, carbon dioxide or ammonia across double bonds or eliminate these to create double
1. Lyases
bonds.

The Isomerases enzymes catalyze the structural shifts present in a molecule, thus causing the
1. Isomerases
change in the shape of the molecule.

1. Ligases The Ligases enzymes are known to charge the catalysis of a ligation process.
 CLASSIFICATION OF ENZYME
Oxidoreductases: These catalyze oxidation and reduction reactions, e.g. pyruvate dehydrogenase, which catalyzes the
oxidation of pyruvate to acetyl coenzyme A.

Transferases: These catalyze the transfer of a chemical group from one compound to another. An example is a
transaminase, which transfers an amino group from one molecule to another.

Hydrolases: They catalyze the hydrolysis of a bond. For example, the enzyme pepsin hydrolyzes peptide bonds in proteins.

Lyases: These catalyze the breakage of bonds without catalysis, e.g. aldolase (an enzyme in glycolysis) catalyzes the
splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Isomerases: They catalyze the formation of an isomer of a compound, example, phosphoglucomutase catalyzes the
conversion of glucose-1-phosphate to glucose-6-phosphate (transfer of a phosphate group from one position to another in
the same compound) in glycogenolysis (conversion of glycogen to glucose for quick release of energy.

Ligases: Ligases catalyze the joining of two molecules. For example, DNA ligase catalyzes the joining of two fragments of
DNA by forming a phosphodiester bond.
 Cofactors
Co-factors are non-proteinous substances that associate with enzymes. A cofactor is essential for the functioning of an
enzyme. An enzyme without a cofactor is called an apoenzyme. An apoenzyme and its cofactor together constitute
the holoenzyme.

There are three kinds of cofactors present in enzymes:

Prosthetic groups: These are cofactors tightly bound to an

enzyme at all times. A fad is a prosthetic group present in
many enzymes.

Coenzyme: A coenzyme is bound to an enzyme only during

catalysis. At all other times, it is detached from the enzyme.
NAD+ is a common coenzyme.
Metal ions: For the catalysis of certain enzymes, a metal ion
is required at the active site to form coordinate bonds. Zn2+ is
a metal ion cofactor used by a number of enzymes.
 ISOENZYMES
Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction but have different enzyme forms and
catalytic efficiencies. Isozymes are usually distinguished by their electrophoretic mobilities. All living systems apparently
require multiple molecular forms of certain enzymes in order to maximize biological capacity.
Isozymes arise from gene duplications and/or different epigenetic modifications of a gene product(s). In this sense, most
of the recombinant enzymes with deletion, insertion, and/or other mutations at the genetic level fall into the category of
isozymes (89). In a restricted definition, “isozymes” are different in genetic origins. An example is the human alkaline
phosphatases which have at least three different genetic origins, i.e., for placental, intestinal, and liver/bone/kidney
enzymes (see Section I,B, Chapter 5). The enzymes that have epigenetic differences due to differential precursor
processings, covalent modifications, and/or tissue distributions are then called isoforms. Examples of isoforms are the
liver/bone/kidney alkaline phosphatases which are encoded by the same gene but differentially modified in a tissue-
specific manner.time
 Mode of Action

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 Factors Affecting Enzyme Activity
Introduction: Biochemical reactions are necessary for growth, repairing damaged
tissues, and obtaining energy and they take place in all living organisms’ bodies.
These reactions are called ‘metabolism’ and they happen all the time in living
organisms. If they stop working, this leads to the death of the organism. All the
reactions that occur in living organisms require high activation energy to take place.
To reduce the cell’s consumption of energy, there is a catalyst to ensure that the
chemical reactions occur rapidly and reduce the activation of energy. This catalyst is
the enzymes.

Enzymes are biological catalysts made up of large protein molecules. They speed up
the chemical reactions inside the cell. The enzyme is made up of a combination of
amino acids which for a chain of polypeptides between each other.
Enzymes are similar to other chemical catalysts. They participate in the reaction
without getting affected. In other words, they speed up the chemical reactions inside
the cells without getting consumed. Enzymes are affected by the hydrogen ion
concentration (pH) and the temperature. Enzymes are highly specific compared to
other catalysts, and each enzyme is specialized for one reactant substance. This
reactant substance is called substrate, and it is specialized for one type of reaction or
a few reactions. Enzymes lower the activation energy required to get the reaction
started. Collectively, these are the most important properties of the enzyme.

There are several factors that affect the speed of an enzyme’s action, such as the
concentration of the enzyme, the concentration of the substrate, temperature,
hydrogen ion concentration (pH), and the presence of inhibitors.
Factor 1. Concentration of Enzyme:
As the concentration of the enzyme is increased, the
velocity of the reaction proportionately increases. This
property is used for determining the activities of serum
enzymes during the diagnosis of diseases.

Factor 2: Concentration of Substrate:

n the presence of a given amount of enzyme, the rate of enzymatic
reaction increases as the substrate concentration increases until a
limiting rate is reached, after which further increase in the substrate
concentration produces no significant change in the reaction rate. At
this point, so much substrate is present that essentially all of the
enzyme active sites have substrate bound to them.
In other words, the enzyme molecules are saturated with substrate.
The excess substrate molecules cannot react until the substrate
already bound to the enzymes has reacted and been released (or been
released without reacting).
Factor 3: Effect of Temperature
•The protein nature of the enzymes makes them extremely sensitive to thermal changes. Enzyme activity
occurs within a narrow range of temperatures compared to ordinary chemical reactions. As you have
seen, each enzyme has a certain temperature at which it is more active. This point is called the optimal
temperature, which ranges between 37 to 40C°.

•The enzyme activity gradually lowers as the temperature rises more than the optimal temperature until it
reaches a certain temperature at which the enzyme activity stops completely due to the change of its
natural composition.

•On the other hand, if the temperature lowers below the optimal temperature, the enzyme activity lowers
until the enzyme reaches a minimum temperature at which the enzyme activity is the least. The enzyme
activity stops completely at 0C°, but if the temperature rises again, then the enzyme gets reactivated
once more.

Factor 4: Effect of pH
•The potential of hydrogen (pH) is the best measurement for determining the concentration of
hydrogen ion (H+)in a solution. It also determines whether the liquid is acidic, basic or neutral.
Generally, all liquids with a pH below 7 are called acids, whereas liquids with a pH above 7 are
called bases or alkalines. Liquids with pH 7 are neutral and equal the acidity of pure water at 25 C°.
You can determine pH of any solution using the pH indicators.

Enzymes are protein substances that contain acidic carboxylic groups (COOH– ) and basic amino
groups (NH2). So, the enzymes are affected by changing the pH value.

Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH. If the
pH is lower or higher than the optimal pH, the enzyme activity decreases until it stops working. For
example, pepsin works at a low pH, i.e, it is highly acidic, while trypsin works at a high pH, i.e, it is
basic. Most enzymes work at neutral pH 7.4.
Factor 4: Effect of pH
Enzymes are protein substances that contain acidic carboxylic groups (COOH– ) and basic amino groups
(NH2). So, the enzymes are affected by changing the pH value.

Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH. If the pH is
lower or higher than the optimal pH, the enzyme activity decreases until it stops working. For example,
pepsin works at a low pH, i.e, it is highly acidic, while trypsin works at a high pH, i.e, it is basic. Most
enzymes work at neutral pH 7.4.

Factor 5: Effect of Activators

•Some of the enzymes require certain inorganic metallic cations, like Mg2+, Mn2+, Zn2+, Ca2+,
Co2+, Cu2+, Na+, K+ etc., for their optimum activity. Rarely, anions are also needed for
enzyme activity, e.g. a chloride ion (CI– ) for amylase.
 Enzyme Marker

Enzymes are highly specialized complex proteins that assist chemical changes in every part of the body. For
example, they help break down food so your body can use it effectively. They also help your blood clot. And
they’re present in every organ and cell in your body. Enzymes are necessary for your body to function properly.

Enzyme markers are blood tests that analyze specific enzyme activity in the body. Some inherited diseases or
conditions can cause these enzymes to stop working or be less efficient. Monitoring the rise or fall of enzyme
levels can aid in the diagnosis of a variety of conditions.

You can test blood for using enzyme markers, or a routine blood test to help uncover abnormalities. In some cases,
you may need to take a test multiple times over the course of several days to measure changes over time.
 Uses of Enzyme

1. Diagnostics uses of enzyme

2. Therapeutic uses of enzyme
3. Lab uses of enzyme
4. Industrial uses of enzyme
 1. Diagnostics Uses of Enzyme
Liver diseases
1. AST(SGOT): 5 to 40 units per liter of serum (the liquid part of the blood).
2. ALT(SGPT): 7 to 56 units per liter of serum
3. ALP(Alkaline phosphatase): 20 to 140 IU/LT, but this can vary from laboratory to laboratory.
4. 5’NT (5'nucleotidase): 2-17 Units per liter.
5. GGT(Gamma- Glutamyl Transpeptidase): 9–48 Units per liter (U/L). (Normal values can vary due to age and sex.)
6. LDH 4,5 (Lactate Dehydrogenase): 100–190 U/L(Older than 12 years)

ALT specific for liver disease

AST(SGOT)/ALT(SGPT) ratio is known as De Ritis ALP
5’NT It are marker of obstructive liver disease
Normal<1 liver disease GGT
Normal >2 Alcoholic liver disease ALP
Normal >3 more alcoholic liver disease Increases level 2-3time to the Normal: Viral hepatitis
Increases level 10-12 to the Normal: Obstructive liver disease
AST/ALT: Slight ↑ Increases level more than 25 to Normal: Malignancy, tumor,
Obstructive jaundice rickets, bone involvement metastasis, Paget’s disease
ALP: ↑ ↑ ↑ ↑
AST/ALT: ↑ ↑ ↑ ↑ ↑
Hepatitis
ALP: ↑
 What does the test result mean?
High ALP usually means that either the liver has been damaged or a condition causing increased bone cell activity is present.
If other liver tests such as bilirubin, aspartate aminotransferase (AST), or alanine aminotransferase (ALT) are also high, usually
the increased ALP is coming from the liver. If GGT or 5'-nucleotidase is also increased, then the high ALP is likely due to liver
disease. If either of these two tests is normal, then the high ALP is likely due to a bone condition. Likewise,
if calcium and/or phosphorus measurements are abnormal, usually the ALP is coming from bone.
If it is not clear from signs and symptoms or from other routine tests whether the high ALP is from liver or bone, then a test for
ALP isoenzymes may be necessary to distinguish between bone and liver ALP.
ALP in liver disease
ALP results are usually evaluated along with other tests for liver disease. In some forms of liver disease, such as hepatitis, ALP is
usually much less elevated than AST and ALT. When the bile ducts are blocked (usually by gallstones, scars from previous
gallstones or surgery, or by cancers), ALP and bilirubin may be increased much more than AST or ALT. ALP may also be
increased in liver cancer.
ALP in bone disease
In some bone diseases, such as Paget's disease, where bones become enlarged and deformed, or in certain cancers that spread to
bone, ALP may be increased.
If a person is being successfully treated for Paget's disease, then ALP levels will decrease or return to normal over time. If
someone with bone or liver cancer responds to treatment, ALP levels should decrease.
Moderately elevated ALP may result from other conditions, such as Hodgkin's lymphoma, congestive heart failure, ulcerative
colitis, and certain bacterial infections.
Low levels of ALP may be seen temporarily after blood transfusions or heart bypass surgery. A deficiency in zinc may cause
decreased levels. A rare genetic disorder of bone metabolism called hypophosphatasia can cause severe, protracted low levels of
ALP. Malnutrition or protein deficiency as well as Wilson disease could also be possible causes for lowered ALP.
Cardiac Marker
CPK-MB (Creatine Phosophokinase): 5-25 international units per liter (UI/L).
LDH-I (Lactate Dehydrogenase): Age Older than 12 years 100–190 U/L
MYGLOBIN: Normal range 0 to 85 nanogram per milliliter (ng/mL)
TROPONIN T: Normal Range Below 0.04 ng/ml
TROPONIN R: The normal range for troponin is between 0 and 0.4 ng/mL
IMA(Ischemia- modified albumin): IMA is produce when albumin comes into contact with the ischemic
tissue.Altering it and making it more resistant to binding metals, IMA is produced continually during ischemia and rises
within 2-3 hours of an ischemic event
Glycogen Phosphorylase BB: More sensitive than the other markers during the first 3-4hours after onset of chest pain

Pancreatic diseases
AMYLASE
LIPASE
Normal values: In a healthy individual, a normal blood amylase level is
around 23-85 units per liter (U/L), although some lab ranges for normal
amylase go up to 140 U/L. A normal lipase level can range from 0-160 U/L
depending on the lab.
BONE DISEASE
ALP Bone isoenzyme pre β:Biochemical Markers of Bone Turnover
in Osteoporosis

6 TYPES OF ALP
α1: Biliary pathology(antitrypsin deficiency)
α2HL:lLiver
Α2 HS: Placenta
PRE β: Bone
ℷ: Intestine
LAP (Leukocyte alkaline phosphatase): leukemoid reaction ( 20 and
100 being considered normal)
CALCITONIN: normal value is less than 10 pg/mL.
Nagao(Germ cell variant)(alkaline phosphatase gene): These
results indicate that the tumor
Kasahara ( fetus intestinal variant): renal cell carcinoma
Regan (Placental varient): Tumour marker for renal cell carcinoma.
 2. THERAPEUTIC USES OF ENZYME

A S P A R Ginase
Rtpa (Recombinant tissue plasminogen): A
Asparginase form of tissue plasminogen activator that is
Acute lymphoblastic leukamia made in the laboratory. It helps dissolve blood
clots and is used to treat heart attacks, strokes,
and clots in the lungs

Alpha1 Antistripsin: used for diagnosis Emphysema

Streptokinase/ Urokinase: break down clots in Papin: Anti Inflammatory agent

some cases of myocardial infarction Pancreatin: Used for diagnosis digestive
Streptodornase: It is used topically on surface disorders
lesions and by instillation in closed body cavities
to remove clotted blood or fibrinous or purulent
accumulations.
 3.LAB USES OF ENZYME

Glucose: Hexokinase- GOD,POD

Urea: Urease
Cholesterol: CHOD-POD ESTIMATION
TG:LIPASE
UA: Urease

Horseradish peroxidase:
ALP:
Recom-DNA: It is used extensively in biochemistry applications.
Rt- Endoneuclease
DNA polymerase:
Nucleosidase:
4. INDUSTRIAL USES OF ENZYME

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