2. Enzymes are proteins that help speed up
chemical reactions in our bodies. Enzymes
are essential for digestion, liver function and
much more. Too much or too little of a certain
enzyme can cause health problems.
Enzymes in our blood can also help
healthcare providers check for injuries and
diseases.
3. What are enzymes?
Enzymes are proteins that help speed up
metabolism, or the chemical reactions in our
bodies. They build some substances and
break others down. All living things have
enzymes.
Our bodies naturally produce enzymes. But
enzymes are also manufactured .
4. What do enzymes do?
One of the most important roles of
enzymes is to aid in digestion. Digestion
is the process of turning the food we eat
into energy. For example, there are
enzymes in our saliva, pancreas,
intestines and stomach.
5. They break down fats, proteins and
carbohydrates. Enzymes use these nutrients
for growth and cell repair.
Enzymes also help with:
Breathing.
Building muscle.
Nerve function.
Ridding our bodies of toxins.
6. What are the different types of enzymes?
There are thousands of individual enzymes in
the body. Each type of enzyme only has one
job. For example, the enzyme sucrase
breaks down a sugar called sucrose. Lactase
breaks down lactose, a kind of sugar found in
milk products.
7. Some of the most common digestive
enzymes are:
Carbohydrase breaks down
carbohydrates into sugars.
Lipase breaks down fats into fatty acids.
Protease breaks down protein into amino
acids.
8. What are the parts of an enzyme?
Each enzyme has an “active site.” This area
has a unique shape. The substance an
enzyme works on is a substrate. The
substrate also has a unique shape. The
enzyme and the substrate must fit together
to work.
9.
10. How do temperature and pH affect enzymes?
Enzymes need the right conditions to work. If
conditions aren’t right, enzymes can change
shape. Then, they no longer fit with
substrates, so they don’t work correctly.
11. Each enzyme has an ideal temperature
and pH:
pH: Enzymes are sensitive to acidity and
alkalinity. They don’t work properly if an
environment is too acidic or basic. For
example, an enzyme in the stomach
called pepsin breaks down proteins. If
your stomach doesn’t have enough acid,
pepsin can’t function optimally.
12. Temperature: Enzymes work best when
your body temperature is normal, about
(37°C). As temperature increases,
enzyme reactions increase. But if the
temperature gets too high, the enzyme
stops working. That’s why a
high fever can disrupt bodily functions.
13. How are enzyme tests used to diagnose
health conditions?
Your healthcare provider can use a variety
of enzyme and protein blood tests to check
for certain health conditions. For
example, elevated liver enzymes could be a
sign of liver disease.
15. erm Meaning
Catalyst
A substance that speeds up a
chemical reaction without
being changed
Enzyme
A biological catalyst (usually
a protein)
Substrate
The reactant molecule that
an enzyme works on
Active site
The part of the enzyme where
the substrate binds
17. The part of the enzyme where the
substrate binds is called the active site.
Here, the enzyme changes shape slightly,
fitting tightly with the substrate and
forming the enzyme/substrate complex.
18.
19. Enzymes help speed up chemical reactions
in the human body. They bind to molecules
and alter them in specific ways. They are
essential for respiration, digesting food,
muscle and nerve function, among
thousands of other roles.
20. BIOMEDICAL IMPORTANCE
Without enzymes, life as we know it would
not be possible.
enzymes occupy central roles in health &
diseases.
Enzymes give information to physicians in
diagnostic & prognosis.
Deficiency may leads to inborn errors of
metabolism.
23. 1. OXIDOREDUCTASES
ENZYMES INVOLVED IN OXIDATION- REDUCTION
REACTIONS.
oxidation reduction
AH2 + B A + BH2
Eg.; alcohol dehydrogenase,
lactate dehydrogenase, xanthin oxidase,
G-6-P dehydrogenase, cytochrom oxidase.
24. 2.TRANSFERASES
ENZYMES THAT CATALYSE THE TRANSFER
OF FUNCTIONAL GROUPS FROM ONE
SUBSTRATE TO ANOTHER.
A – X + B A+B – X
Eg; hexokinase,
transaminases, transmethylases,
phosphorylases.
25. 3. HYDROLASES
ENZYMES THAT BRING ABOUT
HYDROLYSIS OF VARIOUS
COMPOUNDS.
A-B + H2O AH + BOH
Eg; lipase,cholin esterase, acid & alkaline
phosphatases, pepsin, urease.
26. 4. LYASES
ENZYMES SPECIALYSED IN THE ADDITION
OR REMOVAL OF WATER, AMMONIA, CO2 etc.
addition elimination
A-B + X-Y AX – BY
Eg; aldolase, fumarase, histidase.
27. 5.ISOMERASES
ENZYMES INVOLVED IN ALL THE
ISOMERISATION REACTIONS.
Eg; triose phosphate isomerase
retinal isomerase, phosphohexose isomerase,
Epimerase.
28. 6. LIGASES
( Greek: ligate – to bind)
ENZYMES CATALYSING THE SYNTHETIC
REACTIONS WHERE TWO MOLECULES ARE JOINED
TOGETHER & ATP IS USED.
A + B A - B
ATP ADP+Pi
Eg; glutamine synthetase, acetyl CoA carboxilase,
succinate thiokinase, DNA ligase.
29. EC Number
The Enzyme Commission Number (EC
Number) is a numerical classification
scheme for enzymes, based on the
chemical reactions they catalyze.
30.
31.
32. CHEMICAL NATURE &
PROPERTIES OF ENZYMES
ALL THE ENZYMES ARE PROTEIN IN NATURE
WITH LARGE mol.Wt., EXCEPT RIBOZYMES.
33. THE FUNCTIONAL UNIT OF AN ENZYME IS
KNOWN AS HOLOENZYMES, WHICH IS
OFTEN MADE UP OF APOENZYME (THE
PROTEIN PART) AND A COENZYME (THE
NON-PROTEIN PART).
34.
35. holoenzyme apoenzyme+coenzyme
(active enzyme) (protein part) (non-proteinpart)
THE TERM PROSTHETIC GROUP IS USED
WHEN THE NON-PROTEIN MOIETY TIGHTLY
BINDS WITH THE APOENZYME.
MONOMERIC ENZYMES made up of a single
polypeptide.
Eg; ribonuclease, trypsin.
36. Some of the enzymes which posses more than one
polypeptide chain are known as OLIGOMERIC
ENZYMES.
Eg; lactate dehydrogenase, aspertate
transcarbamoylase, etc.
37. When many different enzyme catalyzing
reaction sites are located at different sites of
the same macromolecule, it is called
MULTIENZYME COMPLEX.
Eg; fatty acid synthetase, carbamoyl phosphate
synthetase II, pyruvate decarboxylas, etc.
38. FACTORS EFFECTING ENZYME
ACTIVITY
Enzyme activity can be affected by a variety
of factors, such as temperature, pH, and
concentration. Enzymes work best within
specific temperature and pH ranges, and
sub-optimal conditions can cause an enzyme
to lose its ability to bind to a substrate.
39. FACTORS EFFECTING ENZYME
ACTIVITY
1. CONCENTRATION OF ENZYME
As the concentration of the enzyme is
increased, the velocity of the reaction
proportionately increases.
40.
41. 2. SUBSTRATE CONCENTRATION
Increase in substrate concentration
gradually increases the velocity of enzyme
reaction within the limited range substrate
levels.
A rectangular hyperbola curve is obtained
when velocity is plotted against the
substrate concentration.
42. A. At low substrate concentration, the velocity
of the reaction is directly proportional to
the substrate level.
B. As the substrate is increased, the substrate
concentration is not directly proportional to
the enzyme activity. (1/2 Vmax ) .
C. Further increase in substrate can not make
any effect in reaction velocity, the reaction
is independent of the substrate
concentration. The maximum velocity is
obtained is called ( Vmax ) .
43.
44. MICHAELIS-MENTEN CONSTANT
(Km )
IS DIFINED AS THE SUBSTRATE
CONCENTRATION TO PRODUCE ½ MAXIMUM
VELOCITY IN THE ENZYME CATALYSED
REACTION.
Km is independent of enzyme concentration.
Km is the signature of the enzyme ( Km valve
is thus a constant for an enzyme ).
Km denotes the affinity of enzyme for
substrate.
45. Vmax
V=
Km + [S]
V= measured velocity
Vmax = maximum velocity
S= substrate concentration
Km= michaelis-mentan constant
46. WHAT IS Km VALUE ?
THE SUBSTRATE CONCENTRATION AT HALF
MAXIMAL VELOCITY
47. 3. EFFECT OF TEMPERATURE
VELOCITY OF AN ENZYME REACTION
INCREASES WITH INCREASE IN
TEMPARATURE UP TO A MAXIMUM AND
THEN DECLINES. A BELL SHAPED CURVE IS
USUALLY OBTAINED.
48.
49. . The optimum temperature for most of the
enzymes is between 40°C-45°C. However, a few
enzymes (e.g. venom phosphokinases, muscle
adenylate kinase) are active even at 100°C
50. In general, when the enzymes are
exposed to a temperature above 50°C,
denaturation leading to derangement in
the native (tertiary) structure of the
protein and active site are seen. Majority
of the enzymes become inactive at
higher temperature (above 70°C).
51. 4. EFFECT OF pH
EACH ENZYME HAS AN OPTIMUM pH AT
WHICH THE VELOCITY IS MAXIMUM.
EITHER INCREASE OR DECREASE IN pH
ENZYME ACTIVITY DRASTICALLY
DECREASED.
52.
53. Most of the enzymes of higher organisms
show optimum activity around neutral pH (6-
8). There are, however, many exceptions like
pepsin (1-2), acid phosphatase (4-5) and
alkaline phosphatase (10-11) for optimum pH.
54. 5. EFFECT OF PRODUCT
CONCENTRATION
THE ACCUMULATION OF REACTION
PRODUCTS GENERALLY DECREASES THE
ENZYME VELOCITY.
IT IS ACHIVED THROUGH FEED BACK
MECHANISM.
55. For certain’ enzymes, the products combine
with the active site of enzyme and form a
loose complex and, thus, inhibit the enzyme
activity. In the living system, this type of
inhibition is generally prevented by a quick
removal of products formed.
56. 6. EFFECT OF ACTIVATORS
SOME OF THE ENZYMES REQUIRE CERTAIN
INORGANIC METALLIC CATIONS LIKE Mg,
Mn, Zn, Ca, Co. Cu, etc., FOR THEIR OPTIMUM
ACTIVITY.
57. Metal activated enzymes
Metal activated enzymes are enzymes that
have an increased activity due to the
presence of metal ions. ... However, these
ions are not tightly bound with the enzyme as
in metalloenzymes. The metal can activate
the substrate, thus engage directly with the
activity of the enzyme.
59. 7. EFFECT OF TIME
UNDER IDEAL CONDITIONS (PH,
TEMPERATURE) THE TIME REQUIRED FOR
AN ENZYME REACTION IS LESS.
60. 8.EFFECT OF LIGHT AND RADIATION
EXPOSURE OF ENZYMES TO UV, & X- RAYS
INACTIVATES CERTAIN ENZYMES DUE TO
FORMATION OF PEROXIDES.
Eg; UV rays inhibit salivary amylase.
61. ACTIVE SITE
DEFINITION: THE ACTIVE SITE OF AN ENZYME
IS DEFINED AS THE SMALL REGION OF THE
ENZYME WHERE SUBSTRATE BINDING AND
CATALYSIS OCCURS IS REFFERED TO AS
ACTIVE SITE.
62.
63. SALIENT FEATURES:-
The existence of active site is due to
the tertiary structure of protein
resulting in the three dimensional
native conformation. Loss of native
enzyme structure will result in
derangement of active site.
64. Although all parts are required for keeping
the exact three dimensional structure of the
enzyme, the reaction taking place at active
site. The active site occupies only a small
portion of whole enzyme.
Eg; lysozyme has 129 amino acids . The active
site formed by 35,52,62,63& 101 amino acids.
Active sites are regulated as CLEFTS OR
CERVICES occupying a small region in a big
enzyme molecule.
Active site is FLEXIBLE not RIGID.
65. It possesses SUBSTRATE BINDING SITE &
CATALYTIC SITE.
The substrate binds at active site by non-
covalent bonds. There forces are
hydrophobic in nature.
Enzyme specificity is due to active site.
The amino acids ser, asp., his, lys, arg, tyr, are
repeatedly found at active site.
66. ENZYME INHIBITION
An enzyme inhibitor is a molecule that
binds to an enzyme and decreases
its activity. By binding to enzymes' active
sites, inhibitors reduce the compatibility
of substrate and enzyme and this leads
to the inhibition of Enzyme-Substrate
complexes' formation, preventing the
catalysis of reactions and decreasing (at
times to zero) the amount of product
produced by a reaction.
67. It can be said that as the concentration of
enzyme inhibitors increases, the rate of
enzyme activity decreases, and thus, the
amount of product produced is inversely
proportional to the concentration of
inhibitor molecules. Since blocking an
enzyme's activity can kill a pathogen or
correct a metabolic imbalance, many
drugs are enzyme inhibitors. They are
also used in pesticides.
68. Not all molecules that bind to enzymes
are inhibitors; enzyme activators bind to
enzymes and increase their enzymatic
activity, while enzyme substrates bind
and are converted to products in the
normal catalytic cycle of the enzyme.
69. ENZYME INHIBITION
Enzyme inhibitor is defined as a substance which
binds the enzyme and brings about a decrease in
catalytic activity.
The inhibitor may be organic or inorganic in
nature.
Enzyme inhibitor is a substance which binds with
the enzyme and brings about a decrease in the
catalytic activity of that enzyme
70. Three broad categories of enzyme
inhibition.
1. Reversible inhibition
2. Irreversible inhibition
3. allosteric inhibition
1.REVERSIBLE INHIBITION
The inhibitor binds non-covalently with
enzyme and the enzyme inhibition can be
reversed if the inhibitor is removed. Is
further sub-divided into-
71. i. competitive inhibition
ii. Non- competitive inhibition
iii. Un- competitive inhibition
i. COMPETITIVE INHIBITION
Here the inhibitor (I) closely resembles
the normal substance (S) is regarded as
SUBSTRATE ANALOHGUE.
As long as the inhibitor holds the active
site, the enzyme is not available for the
substrate to bind.
72. During the reaction , ES & EI complexes are
formed.
E + S ES E+P
+
I
EI
Here the reaction velocity is decreased.
Excess substrate abolishes the inhibition.
Km is increased, Vmax unchanged.
74. CLINICALLY USEFUL COMPETITIVE
INHIBITORS
DRUG ENZYME
INHIBITED
CLINICAL USE
1.Allopurinol
2.Dicoumarol
3.Pencillin
4.methotrexat
e
Xanthine oxidase
Vit.K epoxide
reductase
Trans peptidase
FH2- reductase
Gout
Anti-coagulant
Bacteria
cancer
75. ii. NON-COMPETITIVE INHIBITION
The inhibitor binds at site other than active
site.
This binding impairs enzyme function.
No structural resemblance with the
substrate.
Km is unchanged, Vmax islowered.
Increase in substrate concentration can’t
abolish non-competitive inhibition.
76. 3D structure may abolish.
The inhibitor binds with enzyme as well as
the ES complex.
E + S ES E+P
+ +
I I
EI EIS
Heavy metal ions (Ag,Pb,Hg,etc.,) can non-
competitively inhibit the enzyme.
77.
78. iii. UN-COMPETITIVE INHIBITION
The inhibitor doesn't binds with enzyme but only
binds with ES complex.
Un-competitive inhibitor decreases both Km & Vmax .
E + S ES E+P
+
I
ESI
79. Inhibition of placental ALP by phenylalanine
is an Eg. Of un-competitive inhibition.
80. 2. IRREVERSIBLE INHIBITION
The inhibitors binds covalently with the
enzymes and inactivate them.
These inhibitors are usually toxic
substances.
Eg: IODOACETATE is an irreversible inhibitor of
papain & G3PDH.
Iodoacetate combines with –SH groups at
active site & inactivate the enzyme.
81. CYANID inhibits Cytochrome oxidase.
FLUORIDE removes MG & Mn ions & so
inhibit enolase of glycolysis.
DI-ISOPROPYL FLUROPHOSPHATE(DFP) is a
nerve gas developed by Germans during 2nd
world war. It inhibits the enzymes containing
Ser at the active site. Eg; serine protease,
acetyl choline, esterase, ( which has imp.
function in nerve transmission).
82. SUICIDE INHIBITION
IT IS SPECIAL TYPE OF IRREVERSIBLE
INHIBITION.
In this, the structural analogue is converted
to a more effective inhibitor.
Eg; 1. ALLOPURINOL which is oxidized by
xanthine oxidase to ALLOXANTHIN that is a
strong inhibitor of xanthin oxidase.
83. 2. Ornithine difluro methyl
decarboxilase ornithine (DFMO)
Is a inhibitor of trypanosomiasis (sleeping
sickness).
3. 5-flurouracil fluro deoxy uridylate
used in chemotherapy.
84. ENZYME SPECIFICITY
Specificity is a characteristic nature of the
active site.
Enzymes are high specific in their action.
The occurrence of thousands of enzymes is
due to specific nature of enzymes.
It is three types.
1. Stereospecificity
2. Reaction specificity
3. Substrate specificity.
85. 1.Stereo or optical specificity
Stereoisomers are the compounds which
have the same molecular formula , but differ
in their structural configuration.
Human enzymes are specific for L-A.A’s &
D-carbohydrates.
The enzymes act on one isomer.
Eg; L-A.A oxidase & D-A.A oxidase acts on
L&D-A.A’s.
86. Hexokinase acts on D-hexoses
Glucokinase acts on D-glucose.
Amylase acts on α-glycosidic linkages.
87. 2.REACTION SPECIFICITY
The same substrate can undergo different
types of reactions, each catalysed by specific
enzyme.
Amino acids can undergo transamination,
deamination, decarboxilation, etc,.
A.A oxidase keto acid
A.A decarboxilase amine
88. 3. SUBSTRATE SPECIFICITY
It is divided into 3 types
a. Absolute specificity
b. relative specificity
c. broad specificity.
89. a. Absolute substrate specificity
Certain enzymes act only on one substrate.
Eg; glucokinase
glucose glucose-6-phosphate
urease
urea ammonia + co2
90. b. Relative substrate specificity
Enzymes act on structurally related
substances.
Its may depends on BOND or GROUP.
Eg; trypsin hydrolyses peptide linkages
involved in trp., lys.
CHYMOTRYPSIN cleaves phe, tyr, trp.
GLYCOSIDASES cleaves glycosidic bonds.
91. c. Broad specificity
This enzymes act on closely related
substances
Eg; hexokinase acts on glucose. Fructose,
mannose.
92. COENZYMES
DEFINITION : the non-protein, organic, low
mol.wt and dialysable substance associated
with enzyme function is known as coenzyme.
Only protein part of enzyme can’t participate
in catalytic activity.
Many enzymes require coenzymes for
catalysis.
93. The cofactors may organic or inorganic in
nature.
The functional enzyme is referred to as
HOLOENZYME which is made up of a protein
part APOENZYME and non-protein part
COENZYME.
Coenzymes undergo alterations during
enzyme reactions, which are later
regenerated. This is in contrast to the
substrate which is converted to the product.
94. Coenzymes participate in various reactions
involving transfer of atoms or groups like
hydrogen, aldehyd, keto, amino, acyl, methyl,
etc.
Coenzymes play a imp. role in enzyme
function.
The specificity of enzyme is mostly
dependent on the apoenzyme and not on the
coenzyme.
It is heat stable.
95. CO-ENZYMES FROM B-COMPLEX VITAMINS:
Most of coenzymes are the derivatives of
B-complex vitamins.
Eg; TPP, FMN, FAD, NAD+, NADP+, PLP, CoA,
96. NON-VITAMIN CO-ENZYMES :
Not all coenzymes are vitamin derivatives.
There are some other organic substances,
which no relation with vitamins.
Eg; ATP, CDP, UDP, SAM, PAPS.
NUCEOTIDE CO-ENZYMES :
Some enzymes possess nitrogenous base,
sugar and phosphate. Such coenzymes are
known as nucleotide coenzymes.
eg; NAD+, NADP+, FMN, FAD, CoA.
97. MECHANISM OF ENZYME ACTION
Catalysis is prime function of enzyme.
For any chemical reaction to occur, the
reactants have to be in an activated or
transition state.
There are few theories explaining the
MEHANISM OF ACTION OF ENZYMES.
98. Enzyme lower activation energy
Enzymes lower the energy of activation.
Activation energy is defined as the energy
required to convert all molecules of a
reacting substance from the ground state to
the transition state.
The reactants when heated attain the
activation energy.
99. Enzymes don't alter the equilibrium
constant, they only enhance the velocity of
the reaction.
The enzyme reduces the activation energy.
Eg; activation energy needed for
decomposition of H2O2 is 765KJ/mol. In the
presence of platinum, it is 49KJ/mol.and in
the presence of enzyme, the activation
energy is <8KJ/mol.
100. ENZYME SUBSTRATE COMPLEX
FORMATION
The prime requisite for enzyme is that the
substrate (S) must combine with the enzyme
(E) at the active site to form enzyme-
substrate complex (ES) which ultimately
results in the production formation (P).
E+S ES E+P
101. LOCK & KEY MODEL or FISCHER’S
TEMPLATE THEORY
It is the first model proposed to explain an
enzyme catalysed reaction.
ACCORDING TO THIS MODEL,
1. Structural confirmation of enzyme is rigid
2. Active site is complementary to the
substrate.
Which could not explain the flexibility
shown by enzymes.
102. INDUCED FIT THEORY or
KOSHLAND’S MODEL
It is more acceptable and realistic model for
ES complex formation.
AS PER THIS MODEL,
– The active site is not rigid & pre-shaped.
– Substrate binds to a specific part of the enzyme.
– The substrate induces conformational changes
in the enzyme.
103. Allosteric inhibition can also be explained by
the hypothesis of koshland.
It has experimental evidence from the X-ray
diffraction studies.
104. SUBSTRATE STRAIN THEORY
In this model, the substrate is strained due to the
induced confirmation change in the enzyme.
When substrate binds to the preformed active site,
the enzyme induces a strain to the substrate.
The strained substrate leads to the product
formation.
It explains the role of enzyme in increasing the rate
of reaction.
105. REGULATION OF ENZYME ACTIVITY
– The Enzyme activity can be regulated by two
means
Altering the activity of existing enzyme
(fine control)
Altering the concentration of enzymes
(coarse control)
106. REGULATION OF ENZYME ACTIVITY- I
–Allosteric regulation
–Feed back inhibition
–Regulation by covalent modifications
–Activation of latent enzymes
107. REGULATION OF ENZYME ACTIVITY- II
Induction and repression of enzyme
synthesis others
Compartmentation of metabolic pathways.
Substrate concentration
108. ALLOSTERIC REGULATION
Enzymes possess additional sites, known as
allosteric sites such enzymes are known as
allosteric enzymes
Allosteric modulators bind at the allosteric site
regulate the enzyme activity.
Enzyme activity increased when a +ve allosteric
effector binds at the allosteric site
Enzyme activity maybe decreased when a –ve
allosteric effector binds at the allosteric site
109. CLASSES OF ALLOSTERIC
ENZYMES
Depending on Vmax
and Km
K-Class of Enzymes V-Class of Enzymes
Changes Km and not Vmax Changes Vmax and not Km
Similar to competitive group
Non-competitive group
PFK involvedin glycolytic pathway
Acetyl-COA carboxylase involvedin
fatty acid synthesis
110. MECHANISM OF ALLOSTERIC
REGULATION
Most of allosteric enzymes are oligomers
Subunits may be identical or different
Effector binds non-covalently to a site other than the active
site and this regulatory site may be located on a subunit that
it self may not be catalytic subunit
A conformational change occurs in the active site of the
enzyme leading either to activate or inhibition of the enzyme
depending on the type of effector
Accordingly allosteric enzymes in the conformational states
that T (Tense or Taut) and the R (Relaxed)
- T form low affinity form for substrate
- R form high affinity form for substrate
So inhibitor favors T form and activator favors R form
111. TYPE OF EFFECTOR
HOMOTROPIC EFFECTOR
When the substrate itself serves as an effector
it is called as homotropic effector
That is the substrate influences the substrate
binding though allosteric mechanism.
Their effective is always positive.
These enzymes show a sigmoidal curve when
velocity is plotted against substrate
concentration. This contrasts with the
hyperbolic curve charcteristic of enzymes
which follow Michaelis-Menten equation
112. HETEROTROPHIC EFFECTER
• When the effecter is different from the
substrate that is called Heterotrophic
effecter
Heterotrophic interactions are either +ve
or -ve.
113. ENZYMES WITH ALLOSTERIC
EFFECTORS
Features of allosteric inhibition
– Inhibition not a substrate analogue
– Partially reversible when excess substrate
added
– Km is usually increased
– Vmax is usually decreased
– Most allosteric enzymes possess quaternary
structure .
114.
115. ENZYMES WITH ALLOSTERIC
EFFECTORS
Enzyme Metabolic pathway Inhibitor Allosteric
Activator
Hexokinase Glycolysis
Glucose6-
Po4
-
Phosphfruct
okinase
Glycolysis ATP AMP,ADP
Pyruvate
carboxylase
Glucooneogeonesis _ Acetyl CoA
Acetyl CoA
carboxylase
Fatty acid synthesis Palmitate Isocitrate
116. FEED BACK INHIBITION
process of inhibiting the first step by the
final product in a series of enzyme
catalyzed reaction of a metabolic
pathway is referred to as feed back
inhibition
A e1 B e2 C e3 D e4 E
Initial substrate Final substrate
118. When the final product E inhibits the first step it is
called as feed back regulation/ negative feed back
regulation because the product E ultimately stops
its own synthesis
This is an effective mechanism because, it saves
the energy expenditure involved in the synthesis of
a compound which is already available with in the
cell
E.g.
1. Aspartate trans carbamoylase (ATCase)
(pyrimidine metbolism)-CTP inhibits ATCase
2. HMG-COA reductase(Cholesterol biosynthesis)
119. REGULATION BY COVALENT
MODIFICATION
Many enzymes exists in the active and inactive form
which are intercovertible depending upon the
needs of the body
Covalent modification is the change in the activity of
the enzyme by either adding a group to enzyme
protein by covalent bond or removing a group by
cleaving a covalent bond.
Includes,
1. Phosphorylation and dephosphorylation
2. Adenylation and deAdeylation
3. ADP ribosylation,Uridylation ,Methylation
120. • Control of phosphorylation and
dephosphorylation is mainly exhibited by the
hormones like (epinephrine, thyroxine etc)
and an enzyme may be active in the
phosphorylated state or dephosphorelated
state
121. E.g. Glycogen phosphorylase an enzyme involved in
cleavage of glycogen to provide glucose or energy
Phosphorylase exists in the forms
A B
dePhosphorylated form phosphorylated form
inactive form active form
122. Like wise some enzymes are active in the
deposphorylated state and become inactive
when phosphorylated
E.g. Glycogen synthase, Acetyl coa
carboxylase
123. ACTIVATION OF LATENT ENZYMES
Some enzymes exists in latent forms and
latent forms as such are inactive for Eg
enzymes may be synthesized as proenzymes
or zymogens which undergo irreversible
covalent activation by the breakdown of one
or more peptide bonds
E.g. chymotripsinogen, trypsinogen, and
plasminogen are respectively converted to
chymotrypsin, trypsin and plasmin
124. COARSE CONTROL
Modification in the concentration of the
enzyme this is done by regulating the rate of
enzyme synthesis that is Induction and
repression of enzyme synthesis
Induction – Increased synthesis of the
enzyme
Repression – Decreased synthesis of the
enzyme
This is coordinated at the level of gene and is
also mediated mainly through the hormones
125. COARSE CONTROL
E.g. Hormone insulin induces the enzyme
involved in the utilization of glucose
1. Glucokinase
2. Phospho fructokinase
3. Pyruvate kinase
Like wise insulin represses enzyme involved in
glucose synthesis (gluconeogenesis) like
Glucose-6phosphatase
,fructose-1,6biphosphatase
126. COMPARTMENTATION OF
METABOLIC PATHWAYS
Certain substances in the body are both
synthesized and also degraded in the body and
there is no point for simultaneous occurrence
of both
So this can be regulated by compartmentation
that is synthesis pathway occurs in one
organelle and breakdown pathway occurs in
other organelle
E.g. fatty acid synthesis take place in
cytoplasm, fatty acid degradation take place in
mitochondria, more over enzymes of the same
pathway may be present in cytoplasm and
mitochondria
127. Some may be present
only in cytoplasm and
some only in
mitochondria
Eg Heme synthesis
Urea cycle
Gluconeogenesis
For all the these pathways some
Enzyme may be located In
cytoplasm some may be in
mitochondria and so the
intermediates have to be
Shuttled across the
mitochondrial
membrane. This provides a point
where again control can be
exerted
130. CLINICAL APPLICATION OF
ENZYMES
Enzymes are present in all the tissues
It is having different applications clinically
1. Diagnosing a disease
2. To diagnose a congenital disorder
3. Measurement of compounds
4. As therapeutic agents
5. Enzymes linked to insoluble materials are used
as chemical reactors.
6. Most of the drugs are acting by inhibiting the
enzymes
132. Enzyme Tissue affected
Amylase Pancreas
Lipase Pancreas
AST Cardiac , hepatic
ALT Hepatic inflammation
ALP Hepatic obstruction, bone
GGT Hepatic damage
Creatinine kinase Brain, cardiac / skeletal
Ck- MB Necrosed cardiac tissue
Acid phosphatase Prostate
LDH Cellular lysis,non- specific
133. ENZYMES IN DIAGNOSIS
Functional Non- functional
Specific to plasma
Have definite function
Present in high
concentration
Enters plasma from tissues
No function in plasma
Normally the level will be
low
These non- functional enzymes are more
important for clinician to diagnose a disease
134. ENTRY OF TISSUE ENZYMES
INTO PLASMA
They enter
when disease process cause changes in
cell membrane permeability
When cell dies
Since there is gross difference between
intracellular and extra cellular
concentration
135. PATTERN OF ENTRY OF ENZYMES
Greater the tissue damage larger will be the
concentration of enzymes in plasma
Cytosolic enzymes will appear before
mitochondrial enzymes
136. ENZYME LEVELS IN PLASMA
The levels are maintained by the following
factors
1. Rate of release of enzymes into plasma
2. Stability of enzymes in plasma
3. The clearance rate of enzymes by reticulo -
endothelial system (Half –life)
137. WHAT AN ENZYME DOES
E+S ES EP E+P
The enzyme (E) binds substrate (S) and
converts it to product (P).
Note that E recycles.
Overall, S P
138. MEASUREMENT OF ENZYME
CK
CrP + ATP creatine + ADP
pH 9.0
PK
ADP+ phosphoenol pyruvate ATP+ pyruvate
LD
Pyruvate + NADH +H Lactate+ NAD
The change in absorbance produced in
this reaction is a measure of enzyme
activity
139. WE CALL THIS MEASUREMENT AN
ENZYME ASSAY.
Decrease in substrate or
Increase in product
Typically change in absorbance
0 30 s 1 min 2 min 4 min 8 min
141. HOW WE MEASURE ENZYME
ACTIVITY
Choose best conditions. All these variables affect
reaction.
– Temperature
– pH
– enzyme concentration
– substrate concentration
– co-factors
– Inhibitors
Keep everything constant except one variable, and
study that.
142. ENZYME ACTIVITY OFTEN MEASURED AS
COLOUR CHANGE
Time
Absorbance
S P
Increase in absorbance
over time is not linear.
This is because of back-
reaction (or sometimes
the product inhibits).
143. ENZYME ACTIVITY OFTEN MEASURED AS
COLOUR CHANGE
Time
Always measure INITIAL rate
Express this as DA/time (vo).
A
b
s
o
r
b
a
n
c
e
146. Myocardial Infarction
1. Creatine Kinase
First enzyme to rise followin infarction,
CK-MB iso-enzyme is specific
2. Aspartate Amino
Transferase
rise after the rise of CK and return to
normal in 4 - 5 days
3. Lactate Dehydrogenase
Last enxyme to rise. LDH-1 becomes
more than 2 (Flipped Pattern)
(ENZYME PROFILES) IN DISEASES CONT
Bone Disease
Alkaline Phosphates
Marked Elevation in Osteoblastic bone
activity as in rickets.
Labile bone isoenzyme is elevated. Also
in paget’s Disease
147. (Enzyme profiles) in diseases cont
Muscle Disease
1. Creatine Kinase (CK - MM)
Marked increase in Muscle Disease.
CK - MM fraction is elevated.
2. Aspartate amino Transferase
(AST)
Shows an increase in different
types of muscle disease; not
specific
3. Aldolase (ALD)
Earliest enzyme to rise, but not
specific
Prostate Cancer
1. Prostatic Specific
Antigen
Marker for Prostate Cancer. Mild increase in
benign prostate enlargement
2. Acid Phosphatase
Marker for Prostate Cancer. Metastatic bone
disease especially from a
Primary from prostate. Inhibited by L
tartrate.
148. CLINICAL SIGNIFICANCE OF ISO-ENZYMES
Separation of Iso-Enzymes by
1. Heat stability
2. Gel Electrophoresis
3. Immuno Elctrophoresis
4. Lectin Binding Assay
5. Monoclonal Antibodies
149. CREATININE KINASE – TOTAL
•Death of cardiac tissue
•Skeletal Muscle Injury
•Brain Disorders
•To Distinguish CK – MB, CK-MM and CK – BB
can be done.
•If any one increase Total Will increase
153. Algorhythm
Sole Elevation of
ALP
Age of the patient
(Childhood & Adolescence)
High due to bony growth
No need to proceed
No need to proceed
Pregnant
No need to
proceed
Do Liver Enzyme
Gamma GT /
5 Nucleotidase
Normal
Do the Heat
Stability
Test
High
No need to
proceed
154. HOW TO INTERPET? if there is abnormal ALP.
•In moderate increase see the age and sex
of patient. If the patient is in growth spurt
then it is physiological increase due to
bony growth (Osteoblastic Action)
•If the patient is Female and pregnant then
the increase may be due to release from
placenta
•If it is due to Liver other enzymes
pertaining to it can be measured if
normal ------ rule out liver disorder
155. •Do the heat stability test --- Liver
fraction denatures but bony fraction is
stable to heat. So if the levels are still
high after heating it at 57c then the
enzymes may be of bony origin (It will
increase in bony cancer (or) fracture but
the increase occurs in osteoblastic
conditions
•If Measurement is done after heavy fat
meal then mild increase in ALP may be
noticed due to origin from small
intestine.
156. ESTIMATION OF CERTAIN COMPOUNDS (EG.,
GLUCOSE, CHOLESTEROL)
•Earlier 2 decades ago Glucose is estimated
based on their chemical property ,reducing
activity of sugars. But there are other reducing
agents which may come and interfere.
• Hence now a days Glucose is measured by
Glucose Oxidase enzyme. Here the enzyme is so
specific and we may get true value of Glucose in
Plasma.
158. PRINCIPLE OF CHOLESTEROL ESTIMATION
Cholesterol Oxidase
Cholesterol + O2 H2O2 +
Cholest - 4 en- 3 one
Peroxidase
H2O2 H2O +O
O + Colorless Dye colored Dye
which is measured at particular wavelength and it is
directly proportional to cholesterol level in blood
Simple Machines which can give you the results with in few
minutes.
159. ELIZA
• In the modern Clinical Chemistry ELIZA Plays an
important role.
• Especially when we have to measure which are present
in nano grams
E.g. Hormonal Assay and Assay of Proteins present in very
low concentration
• It is Based on the Principle that Antigen is captured by the
specific monoclonal
• Antibody which are tagged by enzymes and its
subsequent reaction with substrate in negligible amounts.
• (Beneficial effect from the wedding of enzyme chemistry
am Immunology)
162. TO CONFIRM THE DIAGNOSIS OF THE CONGENITAL
METABOLIC DISORDERS
They are often due to deficiency of enzymes of one
(or) other metabolic pathway
1.Glucose 6 Phosphatase Von-Gierke’s disease
2.Glucose 6 phosphate dehydrogenase (G6PD)
Hemolytic Anemia
Identification of deficient enzyme confirms the
diagnosis and gene therapy may help to over come
the hurdles in future.
163. ENZYMES AS THERAPEUTIC AGENTS
•Strepto kinase prepared from
streptococcus
•Tissue Plasminogen activator (t PA)
bioengineered in E- Coli Helps in
dissolving the blood clots leading to
myocardial infarction.
They convert the pro-enzyme
plasminogen to plasmin, which cleaves
the insoluble fibrin in to soluble
components.
164. ENZYMES AS THERAPEUTIC AGENTS - CONT
• The Concentration of Enzyme needed for
therapeutic achievement should be high
Since it is rapidly cleaved by Reticulo
Endothelial System.
• This difficulty will be overcome in
future, because the work is now going on
to couple enzyme to solid matrices and
they are implanted in highly perfused
area
