ENZYME

1. ENZYME
Sub: BIOCHEMISTRY AND CLINICAL PATHOLOGY
D. PHARM. Ist Year
Presented by
Dr. ARUN KUMAR
M. Pharm., PDCTM, PhD
Principal
PARMARTH COLLEGE OF PHARMACY, HAPUR

2. Introduction
• Enzymes are biological catalysts synthesized
by living cells that accelerate biochemical
reactions.
• The orderly course of metabolic processes is
only possible because each cell is equipped with
its own genetically determined set of enzymes
• It is only this that allows coordinated sequences
of reactions - metabolic pathways
• Involved in many regulatory mechanisms.
• Almost all enzymes are proteins except
catalytically active ribonucleic acids, the
ribozymes

3. Enzyme
• Enzymes are characterized by three
distinctive features:
• Catalytic Power
– Ability to catalyses biochemical reaction
• Specificity
– A given enzyme is very selective
– Both in the substances with which it interacts
and in the reaction that it catalyzes
• Regulation
– Metabolic inhibitors and activators

4. Enzyme Nomenclature
• Traditionally, enzymes often were named by
adding the suffix –ase to the substrate upon
which they acted
• Ex: phosphatase, urease, catalase, proteases
• Confusion arose from these trivial naming.
• So a new system of nomenclature of enzyme was
developed based on nature of reaction it helps
• Six classes of reactions are recognized
– Within each class are subclasses, and under each
subclass are subsubclasses within which individual
enzymes are listed

5. Classification of Enzyme
• Enzyme are classified on the basis of action it performs
– Oxidoreductases - oxidation–reduction reactions
• Phosphate dehydrogenase
– Transferases - transfer of functional groups
• Methyltransferases, Carboxyltransferases
– Hydrolases - hydrolysis reactions
• Carboxylic ester hydrolases
– Isomerases - isomerization reactions
• Epimerases
– Lyases - addition to double bonds
• Carboxy lyases, Aldehyde lyases
– Ligases - formation of bonds with ATP cleavage
• Amino acid–RNA ligases

6. Intracellular and extracellular enzymes
o Intracellular
o enzymes are synthesized and retained in the cell for the
use of cell itself.
o They are found in the cytoplasm, nucleus, mitochondria
and chloroplast.
Example: Oxydoreductase catalyses biological
oxidation, Enzymes involved in reduction in the
mitochondria.
o Extracellular
o enzymes are synthesized in the cell but secreted from the
cell to work externally.
Example : Digestive enzyme produced by the
pancreas, are not used by the cells in the pancreas but are
transported to the
duodenum

7. Chemical Properties
• Most of enzymes carry out their functions relying
solely on their protein structure
• Many others require non-protein components –
cofactors
– Usually metal ion or non-protein organic part
(Coenzyme)
• Less complex than proteins, tend to be stable to
heat
• Many coenzymes are vitamins or contain vitamins
as part of their structure
• Functional unit of enzyme is known as holoenzyme
– Holenzyme = Apoenzyme + Coenzyme
• Apoenzyme : protein without any catalytic activity

8. • If the enzyme is made of single polypeptide –
monomeric enzyme. Ex: ribonuclease, trypsin
• If the enzyme is made up of more than one
polypeptide – oligomeric enzyme. Ex: lactate
dehydrogenase, aspartate transcarbamoylase
• Multienzyme complex: have multiple enzyme
unit to carry out different reaction in sequence

9. Factors effecting enzyme activity
• The contact between enzyme and substrate is the
most essential pre-requisite for enzyme activity.
• The important factors that influence the enzyme
reaction are
– Concentration of Substrate
– Concentration of Enzyme
– Temperature
– pH
– Product concentration
– Activators
– Time
– Light and radiation

10. Concentration of Substrate:
• The frequency with which molecules
collide is directly proportionate to their
concentrations
Rate ∝ [A]n[B]m, Rate= k[A]n[B]m
– where, nA + mB →P;k =rate constant
• The sum of the molar ratios of the reactants defines
the kinetic order of the reaction
• In the example above, reaction is said to be of (n+m)
order overall but n order with respect to A and m
order with respect to B

11. Michaelis-Menten Constant Km
• Also known as Haldane’s Constant
• Substrate concentration to produce half
maximum velocityin an enzyme catalyst
reaction
• Km is constant and a chracterstic feature of a
given enzyme – strength of Enzyme Substrate
(ES) complex
• Low Km value indicates a strong affinity between
enzyme and substrate
• Majority of Enzyme Km value – 10-5 to 10-2

12. Michaelis-Menten Constant Km
Michaelis-Menten
Reaction

13. Concentration of Enzyme
• Reaction velocity is directly proportional to
concentration of enzyme
• Serum enzyme for diagnosis of disease
– Known volume of serum and substrate taken at
optimum pH and temperature
– Enzyme is assayed in laboratory

14. Temperature
• Velocity of an enzyme reaction increase with the
increase in temperature up to a maximum and then
declines
• Increase in temperature causes increases the
kinetic energy of molecules
• A bell-shaped curve is usually observed
• Temperature coefficient Q10 : increase in enzyme
velocity when the temperature is increased by 100C
• Optimum temperature for most of enzyme – 40 – 45
0C
• Beyond 500C there is denaturation of enzyme

15. pH
• Most intracellular enzymes exhibit optimal activity at
pH values between 6 - 8.
• Balance between enzyme denaturation at high or low
pH and effects on the charged state of the enzyme,
the substrates, or both
• Exception – pepsin (1-2), acid phosphatase (4-
5), alkaline phophatase (10-11)

16. Activators
• Certain metallica cations – Mn, Mg, Zn, Ca, Co,
Cu, Na, K.
• It acts in a various ways
– Combining with substrate
– Formation of E-S metal complex, direct participation
in the reaction and bringing a conformational
changes in enzyme
• There are 2 categories of enzyme requiring
metals for their activity
• Metal activated enzyme:Not tightly held by the enzyme and can
be exchanged easily. Ex: ATPAase (Mg and Ca) and Enolase
• Metalloenzyme: Hold the metal tightly. Ex: alcohol
dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase

17. Other Factor
• Product concentration: Accumulation of
reaction products generally decreases the
enzyme velocity
• Light and radiation: exposure to UV, beta-
gamma and X-rays inactivates certain
enzyme
– Formation of peroxides, ex: UV rays inhibit salivary
amylase activity

18. Mechanism of Enzyme Action:
Active Sites
• The active site of an enzyme is the region that binds
substrates, co-factors and prosthetic groups and
contains residue that helps to hold the substrate.
• Active sites generally occupy less than 5% of the total
surface area of enzyme.
• Active site has a specificshapedue to tertiary
structure of protein.
• A change in the shape of protein affects the
shape of active site and function of the enzyme.

19. Active Sites
This model (above) is an enzyme called
Ribonuclease S, that breaks up RNA molecules. It
has three active sites(arrowed).
Active site:
The active site contains both binding and
catalytic regions. The substrate is drawn to
the enzyme’s surfaceand the substrate
molecule(s) are positioned in a way to
promote a reaction: either joining two
molecules together or splitting up a larger
one.Enzyme molecule:
The complexity of the active
site is what makes each
enzyme so specific (i.e.
precise in terms ofthe
substrate it acts on).
Substrate molecule: Substrate
molecules are the chemicals that an
enzyme acts on. They are drawn
into the cleft of the enzyme.

20. Mechanism of enzyme action
• The catalytic efficiency of enzymes is explained by two
perspectives:
Thermodynamic
changes
Processes at the
active site

21. Thermodynamic changes
• All chemical reactions have energy barriers between
reactants and products.
• The difference in transitional state and substrate is called
activational barrier.

22. Lock and Key Model
Substrate
Enzyme
Products
Symbolic representation of the lock and key model of enzyme action.
1. A substrate is drawn into the active sites of theenzyme.
2. The substrate shape must be compatible with the enzymes active site in order to fit and
be reacted upon.
3. The enzyme modifies the substrate. In this instance the substrateis broken down,
releasing two products.

23. I n d u c e d f i t m o d e l
• More recent studies have revealed that the process is much
more likely to involve an induced fit model(proposed by
DANIAL KOSH LAND in 1958).
• According to this exposure of an enzyme to substrate cause a
change in enzyme, which causes the active site to change it’s
shape to allow enzyme and substrate to bind.

24. In h i b i t i o n
o The prevention of an enzyme processas a result of interaction of
inhibitors with the enzyme.
 INHIBITORS:
Any substance that can diminish the velocity of an enzyme
catalyzed reaction is called an inhibitor.

25. A. REVERSIBLE INHIBITION
o It is an inhibition of enzyme activity in which the
inhibiting molecular entity can associate and dissociate
from the protein‘s bindingsite.
TYPES OF REVERSIBLE INHIBITION
o There are four types:
 Competitive inhibition.
 Uncompetitive inhibition.
 Mixed inhibition.
 Non-competitive inhibition.

26. C o m p e t i t i v e i n h i b i t i o n
• In this type of inhibition, the inhibitors compete with
the substrate for the active site. Formation of E.S
complex is reduced while a new E.I complex is
formed.
Example:
• Statin drugs such as lipitor compete with HMG-
CoA(substrate) and inhibit the active site of HMG
CoA-REDUCTASE (that bring about the catalysis of
cholesterol synthesis).

27. U n c o m p e t i t i v e I n h i b i t i o n
• In this type of inhibition, inhibitor does not compete with the
substrate for the active site of enzyme instead it binds to
another site known as allosteric site.
Example:
• Drugs to treat cases of poisoning by methanol or ethylene
glycol act as uncompetitive inhibitors.
• Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are
uncompetitive inhibitors of liver alcohaldehydrogenase.

28. MI X E D INHIBITION
o In this type of inhibition both E.I and E.S.I
complexes are formed.
o Both complexes are catalytically inactive.
N o n c o m p e t i t i v e
i n h i b i t i o n
o It is a special case of inhibition.
o In this inhibitor has the same affinity for either
enzyme E or the E.S complex.

29. B. Irreversible i n h i b i t i o n
• This type of inhibition involves the covalent attachment of the
inhibitor to the enzyme.
• The catalytic activity of enzyme is completely lost.
• It can only be restored only by synthesizing molecules.
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30. E x a m p l e s o f i r r e v e r s i b l e i n h i b i t i o n
• Aspirin which targets and covalently modifies a
key enzyme involved in inflammation is an
irreversible inhibitor.
• SUICIDE INHIBITION :
It is an unusual type of irreversible inhibition
where the enzyme converts the inhibitor into a
reactive form in its active site.

31. Diagnostic Importance of Enzyme
• Estimation of enzyme activities in biological
fluid is of great clinical importance.
• The enzyme can be divided in 2 groups:
– Plasma Specific or plasma functional enzyme
– Non-plasma specific or plasma non-functional
enzyme

32. Plasma Specific
• Present in the plasma normally and have specific
fucntion
• Their value is higher in plasma than tissue
• They are mainly synthesized in liver and enter the
circulation
• Ex: Lipoprotein lipase, plasmin, thrombin, choline
esterase, ceruloplasmin
• Impairment of liver function or genetic disorder –
leads to enzyme deficiency
• Wilson disease – deficiency of ceruloplasmin

33. Non-plasma specific
• These enzymes are present in the low level
in plasma compared to the tissue
• Estimation of activities of these enzymes serves for
the diagnosis and prognosis of several disease -
markers of disease
• The raised enzyme level may indicate
– Cellular damage
– Increased rate of cell turnover
– Proliferation of cells
– Increased synthesis of enzymes

34. Important Diagnostic Enzymes
• Amylase – Acute pancreatitis
• Serum glutamate pyruvate transferase (SGPT) – liver
disease (hepatitis)
• Serum glutamate oxaloacetate transaminase (SGOT) –
Heart attacks (myocardial infarction)
• Alkaline phosphatase – Rickets, obstructive jaundice
• Acid phophatase – cancer of prostate gland
• Lactate dehydrogenase (LDH) – heart attacks, liver disease
• Creatinine phosphokinase (CPK) – myocardial infarction
• Aldolase – Muscular dystrophy

35. Amylase
• Activity increased in acute pancreatitis
• Normal level – 0.2-1.5 IU/l
• Peak value in 8-12 hrs – onset of disease and
returns to normal in 3-4 days
• Urine analysis
• Serum analysis – chronic pancreatitis, acute
parotitis (mumps) and obstruction of
pancreatic duct

36. Serum glutamate pyruvate transferase
(SGPT)
• Also known as Alanine transaminase (ALT)
• Normal level – 3-4.0 IU/l
• Acute hepatitis of viralor toxic origin
• Jaundice and cirrohosis of liver

37. Serum glutamate oxaloacetate
transaminase (SGOT)
• Also known as Aspartate transaminase
• Normal 4-4.5 IU/l
• Increase in myocardial infarction and also in
liver diseases
• SGPT is more specific for liver disease and
SGOT for MI – SGPT more cytosomal enzyme
while SGOT is cytosol and mitochondria

38. Alkaline phosphatase
• Elevated in bone and liver disease
• Normal : 25-90 IU/l
• Diagnosis for
– Rickets,
– Hyperparathyroidism,
– Carcinoma of bone
– Obstructive jaundice
– Paget’s Disease

39. Acid phophatase
• Normal : 0.5 -4 KA units/dl
• Increased in cancer of prostate gland and
Paget’s Disease
• Good tumor marker
Creatinine phosphokinase (CPK)
• Normal : 10-50 IU/l
• Diagnosis of
– MI - Very early detection
– Muscular dystrophy
– Hypothyroidism
– Alcoholism

40. Lactate dehydrogenase (LDH)
• At least five different isozymes
• Assess the timing and extent of heart damage
due to myocardial infarction MI (heart attack)
– 12 hrs of MI: blood level of total LDH increases, and
there is more LDH2 than LDH1
– 24 hrs of MI: more LDH1 than LDH2
Type Composition Location
LDH1 HHHH Heart and erythrocyte
LDH2 HHHM Heart and erythrocyte
LDH3 HHMM Brain and kidney
LDH4 HMMM Skeletal muscle and liver
LDH5 MMMM Skeletal muscle and liver

41. Aldolase
• Normal ; 2+6 IU/l
• Diagnosis of
– Muscular dystrophy
– Liver disease
– Myocardial infarction
– Myasthenia gravis
– Leukemia