Enzymes : for nurses

1. ENZYMES: FOR NURSES
Mrs. Namita Batra Guin
Associate Professor

2. INTRODUCTION
⦿Are organic molecules that are produced in
the living organisms and increase the rate of
a biochemical reaction without being utilized
in the overall process.
⦿They are non-dialysable, colloidal particles,
which are thermolabile proteins with highly
specific catalytic activity.
⦿They increase the rate of a chemical reaction
by lowering its free energy barrier that
separates a substrate from the product.

3. ⦿ Most of the enzymes are produced with in the
cells of a particular tissue and functions there
only.
⦿ Such enzymes are called as intracellular
enzymes. E.g. enzymes of the glycolysis, citric
acid cycle etc.
⦿ Some enzymes are liberated but functions in
some other tissues such are called as
extracellular enzymes. E.g. proteolytic enzymes
like trypsin, chymotrypsin etc.
⦿

4. IMPORTANCE
• Enzymes play an important role in
Metabolism, Diagnosis, and Therapeutics.
• All biochemical reactions are enzyme
catalyzed in the living organism.
• Level of enzyme in blood are of diagnostic
importance e.g. it is a good indicator in
disease such as myocardial infarction.
• Enzyme can be used therapeutically such as
digestive enzymes.

5. • Enzymes are proteins that increase the rate
of reaction by lowering the energy of
activation
• They catalyze nearly all the chemical
reactions taking place in the cells of the body.
• Not altered or consumed during reaction.
• Reusable

6. PROPERTIES
⦿Proenzymes : most of the intracellular
enzymes are secreted in their active form
called zymase.
⦿While some are secreted in their inactive
form called as proenzyme or zymogen.
⦿Presence of cofactor organic or inorganic are
called as coenzyme.
⦿Holoenzyme consists of proteinaceous part
called apoenzyme while non-proteinaceous as
prosthetic group.

7. PROPERTIES
• The enzyme without its non protein moiety is
termed as apoenzyme and it is inactive.
• Holoenzyme is an active enzyme with its non
protein component.

8. In enzymatic reactions, the substance at the beginning of the
process, on which an enzyme begins it’s action is called
substrate.

9. • Active site:
The area on the enzyme where the
substrate or substrates attach to is
called the active site.
• Enzymes are usually very large proteins
and the active site is just a small region
of the enzyme molecule.

10. • Enzyme molecules contain a special pocket
or cleft called the active sites.

11. CHEMICAL NATURE
⦿All enzymes are protein in nature and have
large molecular weights.
⦿Some RNA molecules called ribozymes, also
have catalyst activity.
⦿Most of the enzymes are simple proteins and
have a single polypeptide chain, there are
various enzymes, which have more than one
catalytic activity and are called multimeric
enzymes.

12. ENZYME SPECIFICITY
⦿Enzymes are highly specific, interacting with
only one or a few substrates and catalyzing
one type of a chemical reactions only.
⦿They exhibit several types of catalytic
specificities, such as stereo-specificity,
reaction specificity etc.
⦿Stereospecificity: many optical isomers are
there. If these isomers act as a substrate for
a particular enzyme. This is called
stereospecificity.

13. ENZYME SPECIFICITY
⦿Reaction specificity: Many substrates can be
used in several reactions but a particular
enzyme will catalyse only one of these
reactions. This is called reaction specificity.
⦿Absolute specificity: An enzyme may have
an absolute specificity for its substrate and
not bind with any other substrate, e.g.
urease, which is specific for urea only.
⦿Relative specificity: An enzyme may be
specific either to a particular group or type
of bond. Thus can be of two types: Group
specificity and Bond specificity.

14. COENZYME AND COFACTORS
⦿Coenzyme is a dialyzable, thermostable, low
molecular weight, organic substance, which
may be referred to as co-substrate or second
substrate.
⦿Coenzymes are derivatives of the B-complex
group of vitamins e.g. TPP (coenzyme form of
vitamin B1), FMN and FAD (derivative of B2),
pyridoxal-5-phosphate (coenzyme form of
vitamin B6)

15. COENZYME AND COFACTORS
⦿Several enzymes require certain metal ions,
called cofactors, for their activity e.g. Mg2+,
Zn2+ etc.
⦿A metal ion may be tightly bound to the
enzyme or loosely associated with it.
⦿When metal ions forms an integral part of the
enzymes are referred to as metaloenzymes.

16. COENZYME AND COFACTORS
–A cofactor is a non-protein chemical
compound that is bound (either
tightly or loosely) to an enzyme and is
required for catalysis.
–Types of Cofactors:
• Coenzymes.
• Prosthetic groups.

17. COENZYME AND COFACTORS
• Coenzyme:
The non-protein component, loosely
bound to apoenzyme by non-covalent
bond.
• Examples : vitamins or compound derived
from vitamins.
• Prosthetic group
The non-protein component, tightly
bound to the apoenzyme by covalent
bonds is called a Prosthetic group.

18. CLASSIFICATION OF ENZYMES
• EC 1. Oxidoreductases
• EC 2. Transferases
• EC 3. Hydrolases
• EC 4. Lyases
• EC 5. Isomerases
• EC 6. Ligases

19. CLASSIFICATION OF ENZYMES
⦿Oxidoreductase:
– Catalyse Oxidation/Reduction Reactions
Act on many chemical groupings to add or
remove hydrogen atoms.
⦿ E.g.- Lactate dehydrogenase.
– Glucose Oxidase.
– Peroxidase.

20. CLASSIFICATION OF ENZYMES
⦿Transferase:
– Transfer a functional groups (e.g. methyl
or phosphate) between donor and
acceptor molecules.
⦿ E.g.
– Transaminases (ALT & AST).
– Phosphotransferases (Kinases).

21. CLASSIFICATION OF ENZYMES
⦿Hydrolase:
– Catalyse the hydrolysis of various bonds Add
water across a bond.
⦿ E.g.
– Protein hydrolyzing enzymes (Peptidases).
– Carbohydrases (Amylase, Maltase, Lactase).

22. CLASSIFICATION OF ENZYMES
⦿Lyases:
– Cleave various bonds by means other than
hydrolysis and oxidation.
– Add Water, Ammonia or Carbon dioxide across
double bonds, or remove these elements to
produce double bonds.
⦿ E.g.
– Fumarase.
– Carbonic anhydrase.

23. CLASSIFICATION OF ENZYMES
⦿Isomerases:
–Catalyse isomerization changes within a
single molecule.
–Carry out many kinds of isomerization:
• L to D isomerizations.
• Mutase reactions (Shifts of chemical
groups).
⦿ E.g.
–Isomerase.
–Mutase.

24. CLASSIFICATION OF ENZYMES
⦿Ligases:
–Join two molecules with covalent bonds
Catalyse reactions in which two chemical
groups are joined (or ligated) with the use
of energy from ATP.
⦿ E.g.
–Acetyl~CoA Carboxylase.
–Glutamine synthetase

25. FACTORS AFFECTING ENZYME
ACTION
⦿TEMPERATURE: they work in narrow range of
temperature, i.e. optimum temperature.
Temperature beyond the optimum
temperature has destructive effects on the
enzyme.
⦿Above temperature of 40˚C, the reaction rate
shows a steep fall. As the protein gets
denatured and looses its biological activity.

26. FACTORS AFFECTING ENZYME
ACTION
⦿pH: Few enzymes functions near neutral pH.
However every enzyme has optimal pH when
it is most effective.
⦿The activity of the enzyme is at peak at
optimum pH.
⦿Change in the pH affects the ionization state
of the enzymes thereby decreasing the
number of active sites.
⦿Extremely high or low pH may denature the
enzyme.
⦿Optimum pH for most enzymes is around 7.0.

27. FACTORS AFFECTING ENZYME
ACTION
⦿Concentration of enzyme: increase in the
concentration of enzymes increases the speed
of the reaction
⦿Concentration of substrate: under
favourable conditions, increase in the
substrate concentration increases the
reaction velocity upto certian limit.

28. FACTORS AFFECTING ENZYME
ACTION
⦿Activators: several enzymes have to get
activated in the presence of minute traces of
some inorganic ions or atoms.
⦿Inhibitors: such as cyanides, fluorides,
carbon mono-oxide inhibits enzymatic
reactions.

29. MODE OF ACTION
⦿LOCK AND KEY MODEL
◼Was proposed by Emil Fischer
◼Enzyme acts on substrate by forming enzyme-
substrate complex.
◼It has active site on its surface in which only
specific type of substrate fits and forms enzyme-
substrate complex.
E+ S E + P

30. MODE OF ACTION
• In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active site
• This is an older model, however, and does not work for all
enzymes

31. ⦿INDUCED FIT MODEL
◼Given by Koshland.
◼Active site of enzyme bears two groups. When
enzyme reacts with the substrate, its binding is
supported by buttressing group to form enzyme
substrate complex.
◼The enzyme substrate complex is then acted upon
by catalytic group which results in the formation
of product and releases enzyme.

33. Enzyme-substrate complex
• Step 1:
• Enzyme and substrate combine to form
complex
• E + S ES
• Enzyme Substrate Complex
+

34. Enzyme-product complex
• Step 2:
• An enzyme-product complex is formed.
• ES EP
ES EPtransition
state

35. Product
• The enzyme and product separate
• EP E + P
The product
is made
Enzyme is
ready
for
another
substrate.
EP

36. ENZYME INHIBITION
⦿Reduction or stoppage of enzyme activity due
to internal or external factors or chemicals is
called enzyme inhibition.
⦿May be reversible or irreversible and
competitive or non-competitive.

37. ENZYME INHIBITION

38. REVERSIBLE INHIBITION
⦿Inhibition that can be overcome through
withdrawal of inhibitor.
⦿Competitive inhibitor competes with the
substrate for the active site of enzyme.
⦿When inhibitor binds with the enzyme, it
prevents the binding of the substrate by
forming enzyme inhibitor complex.
⦿But it is reversible, if substrate conc. is
increased, leading to the removal of
inhibitor.

39. REVERSIBLE INHIBITION

40. REVERSIBLE INHIBITION
⦿Other type is uncompetitive. Where the
inhibitor attaches at the site other than the
active site of substrate.
⦿Both enzyme inhibitor complex and enzyme
substrate inhibitor complex are formed.
⦿ESI may break down to form a product but at
a slower rate.
⦿Thus it decreases the velocity of the reaction.

41. REVERSIBLE INHIBITION

42. REVERSIBLE INHIBITION
⦿Non-competitive reversible inhibition:

43. REVERSIBLE INHIBITION

44. IRREVERSIBLE INHIBITION
⦿In this binding of the inhibitor destroys the
functional group of the enzyme, without
restoring it.
⦿Inhibitor binds at or near to the active site
irreversibly using covalent bond.
⦿Cyanide destroys the activity of cytochrome
oxidase by binding to it.

45. REGULATION OF ENZYME ACTION
⦿ Control of enzyme level: enzyme, substrate and
product themselves regulate the chemical
reaction.
⦿ The product accumulates , it brings the
inhibition of the enzyme. This mechanism is
called as feedback mechanism.
⦿ Control at gene level: gene regulates the
production of enzymes. The gene responsible for
the synthesis of enzyme is activated and
inactivated by the substrate to be metabolized
and end product accumulating in excess
respectively.

46. REGULATION OF ENZYME ACTION
⦿ Allosteric Regulation: Oligomeric enzymes have
two sites: catalytic and allosteric (regulatory
site). These two sites are located apart from each
other on two different subunits of these enzymes.
⦿ They catalyse the committed step that is
generally present in the beginning of the
pathway.
⦿ Certain substances are called, allosteric
modulators or effectors, that bind reversibly to
such enzyme at the allosteric site and regulate its
activity.
⦿ As their interaction brings a conformational
changes at the catalytic site of the enzyme.
⦿ An effector molecule may either activate the
reaction or inhibits it (allosteric inhibition) and is
referred to as allosteric regulator.

47. DIAGNOSTICALLY SIGNIFICANT
ENZYMES
⦿Enzymes are the biological catalysts.
⦿Assay of enzymes present in blood plasma or serum
have been routinely carried out in clinical chemistry
laboratories
⦿Diagnostic enzymes refers to the enzymes that are
used directly or as components of the assay system
for the determination of number of substances
⦿Changes in the concentrations of various
biomolecules are indications of abnormal metabolic
activities, infections, infectious and non-infectious
diseases and inflammatory conditions

48. • Use to detect and quantify certain substances
• As labels in enzyme immuno assay (EIA) system
• There are many alternative techniques which
are routinely used for the diagnosis by clinical
laboratories and include Electrophoresis,
chromatographic techniques,isoelectric focusing
etc

49. LACTATE DEHYDROGENASE
⦿Important enzyme found throughout the body and
involved in glucose metabolism
⦿Tetramer of 2 different subunits(H or M)i.e. Heart or
muscle type.
⦿LDH1 and LDH2 is found predominantly in heart
muscle and in RBCs.Most stable and runs the furthest
in electrophoresis strip.
⦿LDH4 and LDH5 found in liver and skeletal muscle is
the least stable and runs the shortest on
electrophoresis
⦿LDH3 is found in a variety of tissues such as spleen,
lung, endocrine glands and lymph nodes

50. ASPARTATE TRANSAMINASE
⦿These enzymes are found in most tissues through
out the body ,but especially in skeletal muscle,
cardiac muscle, liver and kidney.
⦿It is formally known as glutamate oxaloacetate
transaminase(GOT).
⦿Useful in the diagnosis of myocardial infarction.
Elevated AST levels is indicative of damage to the
myocardium.
⦿Normal range-male 35<U/L,female31<U/L

51. ALANINE TRANSAMINASE
⦿Formally known as glutamate pyruvate
transaminase(GPT)
⦿Found in high concentrations in liver cells and
in much smaller concentrations elsewhere.
⦿Hence a markedly raised plasma activity
indicates a severe liver disease, usually viral
hepatitis or toxic liver necrosis
⦿Normal values-male<45U/L,female <34U/L

52. ALKALINE PHOSPHATASE
⦿High levels are found in liver, bone, placenta and
intestine
⦿Used as a marker of cholestatic liver disease
⦿Highest levels are seen in Paget’s disease. A
moderate rise is observed in osteomalacia.

53. CREATINE KINASE
⦿Also known as creatine phosphatase(CPK)
⦿Mainly found in heart and skeletal muscle and in
brain
⦿It is a dimer made up of 2 types of polypeptide
chains(B orM) in any combination. Thus 3 isoenzymes
are found:BB,the main form of brain,MB-in heart
muscle and diaphragm and MM-both heart and
muscle

54. • Increased plasma activities of CK(MM+MB) results in
severe damage to heart cells.
• Normal range=male-46-171U/L,female-34-145U/L.
•Measurement of CK2 in serum is used in diagnosis of
acute MI, where initial rise is seen within 4-6hrs. Peak
levels are observed after 24hrs which returns to
normal after 48-72hrs.
•Serum CK level also increases in acute cerebrovascular
diseases.

55. ACID PHOSPHATASE
⦿ Maximum activity-pH-5-6
⦿ Found in large amounts in prostate glands and its assay in
plasma has been used in the diagnosis of prostatic
carcinoma
⦿ Also found in liver, red cells, platelets and bone. They are
analyzed by immunoassay techniques or by the actions of
inhibitors. The prostate and red cell forms of enzymes are
inactivated by ethanol, red cell form by formaldehyde, and
the prostate form by L-tartarate
⦿ Normal range-0.1-0.4U/L

56. Α-GLUTAMYLTRANSFERASE(GGT,Α-GT)
• Found in biliary ducts of the liver, in the kidney
and pancreas with the largest amounts being
in kidney
• Also found in hepatocytes where its enzyme
activity can be induced by a number of drugs
and in particular alcohol, thus making GGT,a
useful marker of alcohol induced liver disease
and in particular liver cirrhosis.
• Normal range=male-<55U/L,female-<38U/L

57. AMYLASE
• Found in high concentrations in pancreas and
salivary glands where it is secreted to digest
complex carbohydrates
• Useful in those patients with acute abdominal
pain, to differentiate between patients with
acute pancreatitis and those with appendicitis
• Patients with acute pancreatitis will have high
levels of amylase in their blood
• Normal range=28-100U/L