Enzymes

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 By
Dr. Arshad Mahmood Minhas
MBBS, DTCD, FRSH(LOND), M Phil,
Associate Professor,
Azra Naheed Medical College , Lahore.

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 Most enzymes are Proteins
(tertiary and quaternary
structures)

 Act as Catalyst to
accelerates a reaction

 Not permanently changed in

the process

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Characteristics of Enzymes?
1) speed up chemical reactions
2) are required in minute amounts
3) are highly specific in their action
4) are affected by temperature
5) are affected by pH
6) Some catalyse reversible reactions
7) Some require co-enzymes
8) Are inhibited by inhibitors
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 A. Holoenzyme:
it is an enzyme composed from proteinic part
and non-proteinic part.
 B. Apoenzyme:
it is the proteinic part of the holoenzyme.
 C. Co-factor:
it is the non-proteinic part of the holoenzyme
 D. Metal activated enzyme:
holoenzymes which have a loosely bound
metals on its prosthetic inorganic group.
 E. Co-enzyme:
Non-protein organic substance bound tightly in
usual. specific thermo stable low mol.wt
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F. Metalloenzyme:
enzyme which has tightly bound metals as its
prosthetic group.
G. Isoenzymes:
enzymes which have different structures and
same function.
-Cardiolipin
- is the prosthetic group of the enzyme
cytochrome oxidase.
 Cu++, Fe++ ions are the metalloenzyme of
cytochrome oxidase.

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 Are specific for what
they will catalyze
 Are Reusable
 End in –ase
 -Sucrase
 -Lactase
 -Maltase

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Enzymes work by
weakening bonds
which lowers
activation energy

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The substance
(reactant) an
enzyme acts on is
the substrate
Joins
Substrate Enzyme

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 A restricted region of an enzyme molecule
which binds to the substrate.
Active
Site

Substrate
Enzyme

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 A change in the
shape of an
enzyme’s active
site
 Induced by the
substrate

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 A change in the configuration of an
enzyme’s active site.
 (H+ and ionic bonds are involved).
 Induced by the substrate.

substrate Active Site

Enzyme

induced fit

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 Three factors:

1. Environmental Conditions

2. Cofactors and Coenzymes

3. Enzyme Inhibitors

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 Digestive and coagulation enzymes are secreted
in inactive forms, zymogens or proenzyme.

* Zymogens are activated by trimming of a short

peptide blocking the active site, or by
covalent modification of the zymogen.

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Classification
of Enzymes

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 Enzymes can be classified according to the chemical
reactions they catalyze
1. OXIDOREDUCTASES Oxidation/reduction
(dehydrogenases)
2. TRANSFERASES transfer functional groups (e.g., amino or
phosphate groups) (e.g. kinases)
3. HYDROLASES Hydrolysis
(proteases)
4. LYASES Lysis, generatin double bond
( synthases )
5. ISOMERASES Rearrangement
( e.g. rasemases )
6. LIGASES Ligation requiring ATP
( e.g. synthetases ) 1
OTHLIL 6
Requires two substrates one oxidized and other is
reductase e.g Glucose reductase.
Co-enzyme NADH2 + glucose→ Sorbital + NAD

If reaction takes place in aerobic condition, then called

aerobic dehydrogenase.

Glucose O2 H2 Gluconolactone

Oxidase : they use only O2 as hydrogen acceptor e.g.

Ascorbic acid 1/2 O2 H2O Dehydroascorbic acid

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 B. Transeferase
 Def: enzymes which catalyze the transfer of
C-containing, N- containing and sulfur containing
groups e.g. ALT ( GPT).

 1.Transaminases:
Glutamic acid + pyruvic acid α- ketoglutaric
acid + Alanine
ALT (GPT) enzyme can be found in liver normally,

Significance : help in formation of non-essential

amino-acids :
 e.g. Alanine & Aspartate.

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2. Phospho-tranferases, e.g. Glucose to
glucose-6-po4.

3. Trans-methylases, e.g Nor-adrenaline

to adrenaline

4. Trans-peptidases e.g. benzoic acid + glycine

to hippuic acid.

5. Trans-acylases e.g. Palmiic acid to

Palmityl S-CoA
(transfer of Acyl group)

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Def : enzymes that catalyze the breakdown of a
certain compound by addition of water
molecule e.g.

lactase → glucose + galactose

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Def: enzymes which catalyze the breakdown of
organic compound. Catalyze the removal of
a group from a compound and break it
into two compounds without the addition of
water molecule e.g.

Malic acid Fumaric acid

Fructose by lyase enzyme called ( fructose 1,6

bisphosphate) is cleaved to glyceraldehyde
3-phosphate+ dihydroxyacetone phosphate.

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 Def: enzymes that catalyze the convertion
between isomers.
 The enzyme which catalyze the transforming
between hexoses isomers are called
hexo-isomerase
 e.g.
 Glucose → fructose

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  F . LIGASES

 Def : enzyme which catalyzes the reaction

between two molecules.

 e.g. Co2 + Pyruvic acid → Oxaloacetic-acid.

 Catalyze the reaction of the joining of two
compounds. It requires ATP, GTP e.g.

 Succinic Acid GTP GDP+Pi Succinyl S-CoA

+ CoA-SH
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 1-Some enzymes still retain their old names as
digestion enzymes still use –in pepsin, trypsin.
 2-End in –ase:

Identifies a reacting substance

sucrase – reacts sucrose
lipase - reacts lipid
 3-Describes function of enzyme:

oxidase – catalyzes oxidation

hydrolase – catalyzes hydrolysis
 4-Enzyme named by the name of both the substrate
acted upon and the type of reaction catalyzed.
 e.g. Succinic Dehydrogenase that remove
hydrogen from succinic acid.
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 Mechanism of activation:
* The mechanism of activation may involve unmasking of a
polypeptide chain that may be blocking or masking
the active centers of apoenzyme.
* Proteolytic enzymes are secreted inactive to prevent
digestion of protein of the cell that synthesized
them.
Activation takes place by:
HCL
1- pH – changes: Pepsinogen Pepsin
trypsin
2- Auto-activation: Trypsinogen Trypsin
Entrokinase
3- By other enzymes: Trypsinogen Trypsin

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THANK YOU FOR ATTENTION

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1. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.

2. pH (most like 6 - 8 pH near neutral)

3. Ionic concentration (salt ions)

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 Inorganic substances (zinc, iron) and
vitamins (respectively) are sometimes
need for proper enzymatic activity.

 Example:
Iron must be present in the quaternary
structure - hemoglobin in order for it
to pick up oxygen.

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 Classification of Enzymes
All the enzymes are classified into six groups
.
The name of the group indicates the type of the chemical reaction
catalyzed by the enzymes:

1) Oxidoreductases:
are involved in oxidation and reduction.

2) Transferases:
transfer functional groups (e.g., amino or phosphate groups)
between donors and acceptors.

3) Hydrolases:
transfer water; that is, they catalyze the hydrolysis of a
substrate.

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 4) Lyases:
 add (or remove) the elements of water, ammonia, or
carbon dioxide (CO2) to (or from) double bonds.
mutases, as well as catalyze changes within one
molecule; they include racemases.
5) Isomerases:
 epimerases, cis-trans isomerases, intramolecular
oxidoreductases, intramolecular transferases, and
intramolecular lyases.
6) Ligases (Synthetases):
 join two molecules together at the expense of a
high-energy phosphate bond of adenosine
triphosphate (ATP).

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 Enzymes are proteins with highly specialized catalytic
functions, produced by all living organisms.
* Enzymes are responsible for many essential biochemical
reactions in microorganisms, plants, animals, and human
beings.
* Enzymes are natural protein molecules that act as highly
efficient catalysts in biochemical reactions, that is, they
help a chemical reaction take place quickly and efficiently.
Enzymes are highly specific. *
Enzymes not only work efficiently and rapidly, they are also
biodegradable. *

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 * Enzymes are highly efficient in increasing the reaction
rate of biochemical processes that otherwise proceed
very slowly, or in some cases, not at all.
 * All enzymes are protein in nature , so have the same
properties of proteins as denaturation, precipitation,
electrophoresis ………….etc.
 * They are sensitive to changes in pH and temperature.
 * They function in minute amounts, remain unchanged
chemically during the reaction.

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 THANK YOU

5/1/2012 34
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 1-Some enzymes still retain their old names as
digestion enzymes still use –in pepsin, trypsin.
 2-End in –ase:
Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
 3-Describes function of enzyme:
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
 4-Enzyme named by the name of both the substrate
acted upon and the type of reaction catalyzed.
 e.g. Succinic Dehydrogenase that remove
hydrogen from succinic acid. 3
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 Mechanism of activation:
* The mechanism of activation may involve unmasking
of a polypeptide chain that may be blocking or
masking the active centers of apoenzyme.
* Proteolytic enzymes are secreted inactive to prevent
digestion of protein of the cell that synthesized
them.
Activation takes place by:
HCL
1- pH – changes: Pepsinogen Pepsin
trypsin
2- Auto-activation: Trypsinogen Trypsin
Entrokinase
3- By other enzymes: Trypsinogen Trypsin
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 FACTORS AFFECTING ENZYMES
- The activity of the enzyme is evaluated by measuring the
rate or the velocity of the reaction.
- velocity of the reaction is measured by how many moles of
substrate are converted into products per unit of time.
The factors include the following :
1- PH
2- Temperature
3- Substrate Concentration
4- Enzyme Concentration
5 - Cofactor
6- Enzyme Inhibitors
7- Hormones
8- Radiation & Light
9- Product concentration
10-Enzyme activators
11-Antienzyme & antibodies
12-Concentrations of Co- Factors
 1- Temperature
-Little activity at low temperature.
- Rate increases with temperature.
-Each enzyme has an optimum
temperatures at which the enzyme
acts maximally.(usually 37°C).

- Activity lost with denaturation at high

temperatures.
2- pH
-Each enzyme has an optimum pH at
which the enzyme acts maximally.
- Maximum activity at optimum pH
-Most lose activity in low or high pH.
 3-Substrate Concentration

 As substrate concentration increases,

the rate of reaction increases

 at constant enzyme concentration,

the enzyme eventually becomes
saturated giving maximum activity.

- 4. Enzyme Concentration

 - Increasing enzyme concentration increases the rate of

reaction.
 Further increase in the enzyme can not increase the
velocity of reaction because
 the amount of substrate may not be sufficient to permit
of maximum velocity..
 If during a reaction, an enzyme substrate complex is
formed that dissociates (to reform the free
enzyme and the substrate) or reacts ( to release
the product and regenerate the free enzyme).

 K1 K3
 E +S ES E+P
K2

 “E” is the enzyme, “S” substrate, “P” product,

K1,K2,K3 are rate constant ,

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 Michaelis-Menten Equation
V0 = Vmax[S] / KM + [S]

Rate increase with [S]

Rate levels off as
approach Vmax
More S than active
sites in E
Adding S has no
effect
At V0 = ½ Vmax
[S] = KM
 Michaelis Constant (Km)
 Km is equal to the substrate concentration [S] at
which the reaction is half of its maximum
(1/2Vmax).

( Km is the substrate concentration

at which

v= ½ V max

 It expresses the affinity of the enzyme to its

substrate.
 Low Km means high affinity of the enzyme to
substrate.
 High Km means low affinity of the enzyme to
substrate.
Vmax  Vmax occurs when
enzyme active sites
are saturated with
½ substrate
Vmax
 Km (Michaelis-
Menten constant)
reflects affinity of
Km enzyme for its
substrate

 smaller the Km, the

greater the affinity
an enzyme has for
its substrate
 5-Hormones
e.g. insulin hormone stimulates glucokinase enzyme
while glucocorticoids inhibit it.
Steroid hormones are known to increase the rate of
synthesis of many enzyme.
6-Time
As time is passed the rate of the enzyme catalyzed
reaction diminishes due to:
 Decline of substrate concentration.
 The accumulated product may cause feedback
inhibition of the enzyme.
7-Product concentration
As you increase the product concentration you
decrease the rate of the reaction.
The excess amount of product accumulates
and occupies the active site of the enzyme.
 8-Radiation and light
 Light inhibit most enzyme activity although
some enzymes e.g. amylase is activated by
red or green light.
 Ultraviolet rays and ionized radiations cause
denaturation of most enzymes.
9-Enzyme activators

-Certain inorganic ions e.g. :

 CL¯activate salivary and pancreatic amylase.

 Ca++ activate thrombokinase.
 Bile salts activate lipase enzyme.
 10- Enzyme Inhibitors :
Definition: substances which inhibit (stop) the enzyme
activity.
 It causes a loss of catalytic activity
 Change the protein structure of an enzyme.
 It may be : Non-specific or specific.

11-Antienzymes and antibodies :

Ascaris living in the lumen of intestine secrete
antipepsin and antitrypsin to prevent digestion of
the worm by these enzymes.
Antibodies: if enzyme is injected, the immune system
of the body produces antibodies which will
inactivate these enzymes.
 12- Concentration of cofactors :
 The rate of enzyme reaction is directly
proportional to the concentration of the
cofactors. (metal & minerals).

 Protein molecule that is needed by some

enzymes to help the reaction.

 Tightly bound cofactors are called prosthetic

groups .
 Cofactors that are bound and released easily
are called coenzymes.

 Many vitamins are coenzymes.

THANK YOU
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 Enzyme inhibitors
Definition: Enzymes are protein in nature, can be
inactivated by agents that denature them. The
chemical substances which inhibit the enzyme activity.
 It causes a loss of catalytic activity
 Change the protein structure of an enzyme.
 It may be : Non-specific or specific.
Non-specific inhibitors:
These are inhibitors which exert their effect on all
enzymes or on wide variety of enzymes; e.g. Agents
which precipitate or denaturate proteins.
Specific inhibitors:
These are inhibitors which exert their effect on one
enzyme or
on a small group of related enzymes.
May be competitive or noncompetitive
 * COPETITIVE INHIBITION
The molecule resembling the substrate.
* Can bind to the active site of the enzyme and so it
can form enzyme inhibitor complex E1
* Decreases the affinity of enzymes for substrate.
* Excessive concentrations of substrate will break the
EI complex and then “S” can bind to the
enzyme
* Reversible.
* It depends on Substrate and Inhibitor.
 Examples of competitive inhibitors:
 Allopurinol competes with hypoxanthine for xanthine
oxidase inhibiting the formation of uric acid, so it
is used in treatment of hyperuricemia (gout).
 Dicumarol or Warfarine compete with vitamin K, for
epoxide reductase, so they are used to reduce
prothrombin synthesis.
 Statins (e.g. atorvastatin) competes with HMGCoA for
its reductase,so, it inhibits cholesterol synthesis.
 Methotrexate competes with dihydrofolic acid for
dihydrofolate reductase, so, it inhibits DNA synthesis
and used in treatment of cancers.

 EFECT OF ENZYME INHIBITOR

 Competitive Inhibitor

Succinate Dehydrogenase

NON- competitive inhibitors

 Does not have a structure like substrate
 Binds to the enzyme but not active site
 Changes the shape of enzyme and active site Substrate
cannot fit altered active site
 No reaction occurs
 Effect is not reversed by adding substrate
  Allosteric inhibitors

 Allosteric inhibitors are low-molecular weight

substances, they regulate the enzyme activity.
 The inhibitor is not similar in structure to the
substrate.
 it is bound to apoenzyme at sites far from the active
site this site is called allosteric site.
 It is not reversable by increasing the concentration
of the substrate.
 When Allosteric inhibitor is consumed, the activity of
the enzyme is regained, so Allosteric inhibition is
reversible. e.g. glucose-6-phosphate is
allosteric inhibitor for hexokinase enzyme.
-
 Enzyme activity may increase or decrease after the
covalent addition of a chemical group.

 1.Phosphorylation affects many enzymes :

Pyruvate dehydrogenase7 glycogen synthaseare
inhibited by phosphorylation, while glycogen
phosphorylase is activated.
2. Phosphatases :
that remove the phosphate groups alter the activities
of these enzymes.

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 Regulation by Protein-Protein interactions :

 Proteins can bind to enzymes, altering their activities

e.g.
regulatory subunits inhibit the activity of protein
kinase A.

 When these regulatory subunits bind cyclic AMP &

are released from the enzyme, the catalytic
subunits become active.

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  ISOENZYMES
 These are the multiple forms of an enzyme that differ
in structure and properties but they catalyse the
same reaction.
 These are different isomers of the same enzyme which
differ by having a different electrophoretic mobility
and different tissue source. Each of these is called
isoenzyme, and all of them have the same catalytic
activity
 e.g. lactate dehydrogenase (LDH) and
creatin kinase (CK).
 Creatine kinase (CK)
1) consisting of two subunits can be present as :
two distinct molecular forms brain type (B) and
muscle type (M).
Thus, three isozymes are possible CK-MM, CK-BB, and
CK-MB.
CK-MB is normally present in trace amounts only in the
myocardium. Elevation of CK-MB levels to greater
than 6% of the total CK is diagnostic of a
myocardial infarction.
1) CPK is made of three slightly different substances:
CPK-1 (also called CPK-BB) is found mostly in the
brain and lungs.
CPK-2 (CPK-MB) is found mostly in the heart.
CPK-3 (CPK-MM) is found mostly in skeletal muscle.
 2) Lactate dehydrogenase (LDH)
 is a tetramer of four subunits which can be present as
two distinct molecular forms.
 Type H is found primarily in heart, and
 Type M is found primarily in muscle or liver.

 Five isozmes of LDH composed of different

combinations of these subunits are possible:
M4, M3H, M2H2, MH3 and H4.

 In normal serum the H3M isozyme is present.

 In a myocardial infarction,the serum levels of the H-

containing isozymes,particularly H4, are elevated.
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  LDH exists in 5 isoenzymes ,
which differ s lightly in subunits
LDH-1, HHHH is found primarily in heart muscle and
red blood cells.
LDH-2 , HHHM is concentrated in white blood cells.
LDH-3 , HHMM is highest in the lung.
LDH-4 HMMM is highest in the kidney, placenta, and
pancreas.
LDH-5 , MMMM is highest in the liver and skeletal
muscle.
•

18hr 48-72hr

• SGOT(AST) 6-8 hrs. 24hr 3-7 days

• Treponin 4-12 hrs 5-10 days

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 Examples for medical important enzymes :

Transaminases :

 1- Glutamic pyruvic transaminase (SGPT) ALT increased

in any disease causing damage or destruction to liver
cells e.g. infective hepatitis.
 2- Glutamic oxaloactic transaminase SGOT (AST) :
is elevated in damage of heart e.g. coronary
thrombosis and liver disease.

 Lactic Acid Dehydrogenase (LDH) is elevated in

cases of myocardial infarction.
 Trypsin: also increased in pancreatic diseases.
 Cholinesterase: is lowered in exposure to
insecticides and is elevated in conditions of
active heamopoiesis
 Serum alkaline phosphatase : it is elevated in :
1- Liver diseases as obstructive jaundice.
2- Bone diseases: rickets, osteogenic sarcoma
and Paget's disease.
 3- Hyperparathyroldism and malignancy.

 Serum Acid Phosphatase is elevated in cancer

prostate.
 Lipase: it is elevated in acute pancreatitis and
pancreatic carcinoma.
 Amylase: it is elevated in parotitis, acute
pancreatitis and pancreatic carcinoma.
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 Therapeutic Uses of ENZYMES

 Streptokinase & urokinase AC.MI, DVT, PUL.Embolism

 L-Asparginase Ac.Leukemia,lymphomas
 Digestive Enzymes Cyctic fibrosis,Ch.pancreatitis
 Chymotrypsin Inflammation,oedema injury
 Used locally, Hyaluronidase Traumatic Inflam;operations

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 Enzymes for disease diagnosis

Serum enzymes Diseases

(elevated)
Amylase Acute pancreatitis

Serum glutamate pyruvate Liver diseases (hepatitis)

transaminase (SGPT)
Serum glutamate oxaloacetate Heart attack (myocardial infarction)
transaminase (SGOT)
Alkaline phosphatase Rickets, obstructive jaundice

Acid phosphatase Cancer of prostate gland

Lactate dehydrogenase (LDH) Heart attack, liver diseases

γ-glutamyl transpeptidase (GGT) Alcoholism

5’-nucleotidase Hepatitis

Aldolase Muscular dystrophy

 Serum Acid Phosphatase is elevated in cancer prostate.
 1. Enzymes estimation in serum and body
fluids for diagnosis and prognosis.e.g
ALT,LDH, AST
 2. Enzymes used as laboratory reagent.e.g
 Glucose oxidase Glucose
 Cholestrol oxidase Cholestrol
 Lipase Triglycerides
 Uricase Uric acid
 Urease Urea
 3. Therapeutic uses of enzymes.
 Name Availability Mech.of Action
Indications Vial 750- Increases amount of
Streptokinas Ac.Myocar .Infarction
e 150,000 Plasmin Ac.thrombosis of Art.
Urokinase IU Pul.Embolism
50,000- DVT
500,000 I
U
L- As lunase Certain Tumour cells Ac. Leukemia
Asparginase require Malignant lymphoma
L-aspargine for growth
Digestive As Syrup Replacement therapy in Cystic
Enz. & tablets pancreatic insufficiency fibrosis,Chr.pancreatitis,
Amylase,lipa following pancreatectomy
se& protease
α- Sublingua Mucolytic & proteolytic Management of inflam-
chymotrypsi l tablets activity matory oedema,post-
n surg. infections and
dental procedures
Serrato - 5 mg Fibrinolytic activity,high Effectivein inflammation
peptidase tablet brady-kinin decomposing after trauma
activity & potent injury,operation
caeinolytic activity subconjuctival bleeding
Enzyme Reaction Use Enzyme Reaction Use
Amylase Starch Diagestive Rhodanase Degradation Cyanide
hydrolysis disorders of cyanide poisoing
Collagena Collagen Skin ulcers RNase RNA Antiviral RNA
se hydrolysis digestion hydrolysis

Ficin, Protein Deworming,  lactamase PencillinPen Pencillin

Papain, hydrolysis digestive - cilloate allergy
Proteinase disorders,nar Streptokinase Plasminogen Blood clot
cotic tissue /urokinase plasmin dissolving
removal
uricase UAallatoin Gout
Glutamina GlnGlu Leukemia Hyaluronidas ?HUA Viral
se e hydrolysis infection
Aspargina AsnAsp Leukemia SOD 2O2+2H+H antioxidant/ant
se 2O2+O2 iinflammatory

Lysozyme Bacterial CW As urease Urea Hyperureamia

hydrolysis Antibiotics hydrolysis
Bilirubin Bilirubin hyperbilirubi Arginase Arginine Cancer
oxidase hydroxylase nemia hydrolysis
 Organ Enzymes Diseases

Liver ALP,GDH,SGOT,SGPT, Viral/toxic hepatitis

Glutamyl Tranferasess, or cirrhosis,
Glutathione S Tranferases, myocardial
infraction,jaundice,
Heart Creatne Kinases,LDH, Myocardial
AST/ALT, Infraction,skelital
muscle disorders.
Other Amylases,Aldolases,TG GIT disorders,
diseases Lipases,CK,ALP,ACP,Choline jaundice,bone
Esterases,HMG CO A diseases, rickets,
Reductases,Xanthine
Oxidases,
 Diseases Enzyme Disease Enzyme

Gout (X linked PRPP Synthase Tay-Sachs disease hexosaminidase A

recessive)
Krabbe's disease b-galactosidase
Lesch Nyhan Syndrom hypoxanthine-
guanine-
Phosphoribosyl Niemann-Pick sphingomyelinase
transferase disease

Cystic fibrosis DNase Farber's disease ceramidase

Alcaptonuria Homogentisate Fabry's disease a-galactosidase

oxidase
Alcaptonuria homogentisate
Maple syrup urine Branched-chain oxidase
disease ketoacid
(various forms dehydrogenase Gauchers desease b-glucosidase

Immunoinsufficiency(a Adenosinedeamin
Lactose intolerance Lactase
utosomal Recessive) ase
 Drug/Inhibitor Enzyme Disease
Mevinolin, Copactin, HMG CoA Reductase Hypercholesteremia
Monacolk
Penicillins & antibiotics Peptidyl transferase, Infections after
RNA polmerases,DNA Surgery,Other
Polymerases etc. infectious diseases
Allopurine Xanthine Oxidases Gout, Hyperureamia
HIV Protease HIV Proteases AIDS
Inhibitors
Methotrexate Folate reductase Cancer,infections
Ethanol Alcohol Methanol poisoning,
Dehydrogenase
PRODRUG DRUG ENZYME
Methotrexate phe. methotrexate Carboxy peptidase A
Etoposide phosphate Etoposide ALP
N bis 2-chloroehyl Benzoic acid mustard Carboxypeptidase-G2
aminobezoyl glu.
Mtomycin phosphate mitomycin ALP
Aniline mustard Aniline mustar Glucuronidase
glucuronide
5-flurrocytosine 5-flurouracinl Cytosine deaminase

2 L amino acyl mmethotrexate aminopeptidase

methotrexate
Drugs
While some enzyme inhibitors are poisonous, others
are beneficial to life.
Penicillin acts as an enzyme inhibitor for transpeptide, a
substance that bacteria need to build their cell walls.
If the cell wall is lacking, osmotic pressure causes the
bacterial cell to burst and die.
However, new strains of bacteria have developed an
enzyme, penicillinase, that inactivates penicillin.
To destroy these new strains, synthetically modified
penicillins have been prepared so that this antibiotic
remains effective.
 ENZYMES
.
CLINICAL SIGNIFICANCE OF RELATIVE AMOUNT OF LDH

Condition Isoenzyme Pattern

Moderate elevation of LDH1;
Myocardial Infarction Slight elevation of LDH2
Large elevation of LDH5;
Acute Hepatitis Moderate elevation of LDH4

Muscular Dystrophy Elevation of LDH1, LDH2, LDH3

Megaloblastic Anemia Large elevation of LDH1

Sickle-cell Anemia Moderate elevation of LDH1, LDH2

Arthritis with Joint effusions Elevation of LDH5

82
Clinical Significance of
L.F.T’s
By

Dr Arshad M Minhas
M.B.B.S, DTCD,FRSH(LOND), MPhil.
Assistant Professor
ALLAMA IQBAL MEDICAL
College,Lhr.
 Normal Liver Functions

1. Synthetic 2. Metabolic 3. Detoxification/ 4. Storage

Plasma Proteins Carbohydrates Excretion Iron
Coagulation Bilirubin
Factors Proteins Glycogen
Fats Drugs Blood
Primary Bile Acids
Toxins
Bile Salts
Amino Acid
Cholesterol
Steroid Hormones
1. Carbohydrate Metabolism
 Storage of glycogen
 Conversion of glycogen  glucose
 Conversion of monosacharides to glucose e.g. (frucotose,
galactose).

2. Protein Metabolism
 Breakdown of a.a. e.g. deamination
 Formation of plasma proteins
 Albumin
 Globulin
 Clotting factors
 Formation of urea
3. Fat Metabolism
 Formation of lipoproteins
 Formation of ketone bodies
 Synthesis of cholesterol
 Formation of phospholipids
 Saturation, denaturation, shortening / lengthening of fatty acid chain.
4. Secretion of Bile
 Formed during waking 23ml/hr or 1-2 litters/day.Ultimately stored in G.B.
5. Metabolism OF Bile Pigment
 Unconjugated (albumin bound) bilirubin is conjugated. 250-350 mg of
bilirubin/day. 10-20 million of RBCs lysed/hr. 6 gms of Hb is lysed/day (1
gm of Hb  35 mg of bilirubin )
6. Metabolism of Bile Salts 0.8 gms of bile salts synthesized/day
 Cholic acid Chenodeoxycholic aicd
7. Formation of blood cells
 In intrauterine life
 Has potential in adult life

8. Destruction of RBCs
 Kupfer cells

9. Storage Funciton
 Iron
 Vitamin A, D, K, B12, follic acid.
 Stores blood to regulate blood volume if some one drinks lot
of H2O
10. Detoxification functions
 Conjugation
◦ Glucoronic acid
◦ Sulphates
 Hydrolysis
 Reduction
 Oxidation
11. Enzymes
1. Cytosolic ALT, AST 20% ,
 LDH
2. Membranous (cytoplasmic and mitochondrial)
◦ AST 80%
◦ ALK PO4ase
◦ GGT (gamma glutamyl transferase)
In our clinical practice minimum liver function tests
which are recommended clinically to evaluate clear
functional picture of the liver.
Parameter Reference range
Total bilirubin 0.1 - 1.0 mg%
Direct bilirubin Up to 1.0 mg%
SGPT (ALT) M- upto 42 F-upto 35 IU

SGOT (AST) Upto 35 IU

Alkaline phosphatase 80 – 300 IU
GGT (gamma glutamyl 10 – 50 IU/lit
transferase)
Total protein 6.5 – 8.5 gms%
Albumin 3.5 – 5.5 gms%
Globulins 1.8 – 3.2 gms%
A/G ratio 1.2 – 2.2
PT (Prothrombin time) 11 – 13.5 sec
 Explanations of (LFTs) Parameters

• Bilirubin
Hb 120 Heme + globin + iron
days

Heme oxygenase
system

Biliverdin
Liver enzymes
 Liver enzymes are normally located in the hepatocytes. They
only come out if the hepatocytes are damaged and their activity
in the plasma is increased.
 We divide the enzymes into two main classes

Cytosolic
◦ ALT
◦ AST 20%
◦ LDH
Membranous (cytoplasmic and mitochondrial)
Obstructive
◦ AST 80% Jaundice
◦ ALK PO4ase
◦ GGT
(gamma glutamyl transferase)
Liver enzymes
 A rise in the plasma transminase activities is a sensitive
indicator of damage of cytoplasmic and mitochondrial
membranes.
Liver cells contain more AST than ALT, but ALT is confined
to the cytoplasm in which its concentration is higher than
that of AST.
 Increased plasma activity of ALT is highly specific
parameter for the evaluation of hepatic cell damage.
In infective conditions.
 like viral hepatitis B,C cytoplasmic membrane sustain the
main damage , leakage of the enzymes cause relatively
greater increase in plasma ALT than AST.
Liver enzymes
In infiltrative conditions.
 There is damage to both cytoplasmic and mitochondrial
membrane , there is proportionally greater increase in the
AST activity than ALT.

1- ALT.(SGPT) Alanine Transaminase

 Male up to 42 IU and Female up to 35 IU

 Most specific test B/C ALT is primarily found in liver.

 Results are expresses as (ULN) upper limit normal. i e
Twice the upper limit normal.
2-AST(SGOT)Aspartate transaminase. Normal value (up to 35
IU)
 Not only found in liver but also in Heart, Muscle, RBCs,
Brain, and Kidney.
Liver enzymes
ALP Alkaline phosphatase (Normal value 80 to 300 IU)
Enzyme which line the biliary ducts .
As ALP is also present in Bones, Placenta and Intestine.
This is also not very specific test , but when its level is
raised another test serum GGT is recommended to
confirm whether increased level of ALP is due to biliary
tract obstruction or by the Bone diseases.
GGT Gamma Glutamyl Transferase.
Is an enzyme present in the ER of Hepatobiliary Epithelial
Cells. It is involved in the transfer of Amino Acids across
the cell membrane.
.GGT is increased in obstructive jaundice
As the ER proliferate in response to prolonged intake of
Alcohol or Drugs like phenobarbitone, phenytoin,
 Significance & Importance of LFTs.

1- Diagnosis of Liver Disease.

2- To know the Severity of the Disease.

3- To differentiate between different types of Diseases.

4- Follow – Up ( Disease).
 ENZYME STRUCTURE, SPECIFICITY AND ACTION
Structure of the Enzyme
* Enzymes are proteins, and their function is determined
by their complex structure. The reaction takes
place in a small part of the enzyme called the
active site, while the rest of the protein acts as
"scaffolding".
* The enzyme is tertiary or quaternary structure that has
spatial configuration.
This makes the enzyme specific for one reaction only, as
other molecules won't fit into the active site – their
shape is wrong.
* It has pockets on its surface. Each pocket has its own
function.
 The catalytic or active site.
 The allosteric site.
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 1) The catalytic or active site:
- It is the site at which the substrate binds to the enzyme.
- It should be fit to the substrate (fitness is made by the
tertiary structure of the enzyme molecule).
- Any factor affecting this structure will alter the fitness
and formation of enzyme-substrate complex.
2) The Allosteric site:
- It is a site for fitting of a small molecule whose binding
alters the affinity of the catalytic site to the
substrate.
- This small molecule is called allosteric modifier for binding
of the substrate:
Stimulatory (making it more fit)
Inhibitory (making the catalytic site unfit
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Enzyme Action
- An enzyme binds a substrate in a region called the
active site
Only certain substrates can fit the active site
forming enzyme substrate complex :
- The active site contain specific groups of Amino acid
help substrate bind.
- Enzyme-substrate complex decomposes, giving rise
to free enzyme and products of the reaction.
E+S ES E+ P
Mode of enzyme action
• The reactants should raise their energy levels to reach
a transition state.
• The transition state represents the energy barrier
between the reactants and products.
• This energy is known as energy of activation.
• The enzyme makes the reaction needs lower activation
energy
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How do enzymes work?
- There are three parts to our thinking about enzyme
catalysis.
- They each describe different aspects of the same
process, and you should know about each of them.
1. Reaction Mechanism
In any chemical reaction, a substrate (S) is converted
into a product (P). -
- In an enzyme-catalysed reaction, the substrate first
binds to the active site of the enzyme to form an
enzyme-substrate (ES) complex, then the
substrate is converted into product whilst
attached to the enzyme, and finally the product is
released, thus allowing the enzyme to start all
over again.
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2. Molecular Geometry:
- The substrate molecule is complementary in shape to that of
the active site. It was thought that the substrate
exactly fitted into the active site of the enzyme
molecule like a key fitting into a lock (the now
discredited ‘lock and key’ theory).
- It is now known that the substrate and the active site both
change shape when the enzyme-substrate complex is
formed, bending (and thus weakening) the target bonds.

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3. Energy Changes:
- The way enzymes work can also be shown by looking at
the energy changes during a chemical reaction.
- In a reaction where the product has a lower energy than
the substrate, the substrate naturally turns into
product (i.e. the equilibrium lies in the direction of
the product).
- Before it can change into product, the substrate must
overcome an "energy barrier" called the activation
energy.
- The larger the activation energy is, the slower the
reaction will be. This is because only a few
substrate molecules will have sufficient energy to
overcome the activation energy barrier

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 Enzyme specificity
The specificity of an enzyme is determined by:
- The functional groups of the substrate (or product).
- The functional groups of the enzyme and its cofactors.
- The physical proximity of these various functional groups.
Two theories have been proposed to explain the
specificity of enzyme action
The active site of the enzyme is complementary in The lock
and key theory:
A) conformation to the substrate, so that enzyme and
substrate "recognize" one another.

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E S complex E+P E+ S

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B)
- The active site of the enzyme is flexible, not rigid
- When the active site identifies the substrate it
brings a change in the active site shape so it can
accommodate the substrate.
- It has a shape complementary to that of the
substrate only after the substrate is bound to
the enzyme.
-This model similar to the rubber gloves.

E+S ES complex E+P

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There are 5 types of specificity:
1) Absolute specificity :
e.g. urease enzyme acts on urea, uricase enzyme acts on
uric acid and arginase enzyme acts on arginine.

2) Relative Specificity:
In this type, the enzyme acts on a group of closely
related substrates i.e. which are similar in
structure and posses the same type of bonds e.g.
Lipase catalyzes the process of hydrolysis of ester
linkage present in triglycerides containing
different types of fatty acids.

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3) Stereo
The enzyme works on one of two isomers e.g.: L – Amino
acid oxidase acts cn L – amino acids only. &
Enzymes of glycolysis act on D-sugars only.
Exception:
There is only one exception which is racemase enzyme
catalyses reversible interconversion of D and L
isomers.
4) Dual specificity:
- The enzyme acts on 2 different substrates e.g.:
Xanthine oxidase can oxidize hypoxanthine and
xanthine to uric acid.
Isocitric acid dehydrogenase acting on isocitric acid
causing dehydrogenation and decarboxylation.
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5) Group
-The enzyme shows specificity not only to the bond
and its position but also towards the chemical
groups or atoms surrounding this bond e.g.
 carboxy peptidase acts on the free carboxyl
end of polypeptide chain .
 amino peptidase acts on the free amino end of
polypeptide chain .

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