Applications of Enzymes

Dr. Javed Anver Qureshi

Professor of Biochemistry,
Institute of Molecular Biology and
Biochemistry (IMBB)
The University of Lahore
 Enzymes

Introduction to Enzyme as
The Properties
Enzymology Biological
of Enzymes
Catalysts

Factors
Enzyme affecting
Enzyme kinetics
classification enzyme activity

Regulation of Applications of
Enzyme
enzyme Enzyme
Inhibition
activity.
 Applications / Uses of enzymes
Medical uses : Medicine
Commercial , industrial uses
Research
 MEASURING ENZYMATIC ACTIVITY

Enzymes are never expressed in terms of their concentration (as

mg or μg etc.), but are expressed only as activities.

Enzyme activity = moles of substrate converted to product per

unit time.

The rate of appearance of product or the rate of disappearance

of substrate
Test the absorbance: spectrophotometer 

Measuring Enzymatic Rates ideally done with a system where

the product or substrate absorb a particular wavelength of light
this depends on enzyme reaction can be monitored with a
spectrophotometer by measuring either  the appearance of
product or  disappearance of substrate
 IMPORTANCE OF ENZYME’S ACTIVITY

In human body the enzymes involved in all essential biological
reaction for life such as DNA replication and transcription,
protein synthesis, metabolism and signal transduction 

In industry enzymes play role in industrial products and

processes, for example, within the detergent, textile and starch
industries. 

In food processing and storage for producing some foods like

cheese, and also some enzyme cause deteriorative actions
and enzymic browning
Enzyme kinetics ( Activity )

• The rate of a chemical reaction is described by the number of

molecules of reactant(s) that are converted into product(s) in a
specified time period.

• International unit – One IU is defined as the activity of the

enzyme which transforms one micro mole of substrate in to
products per minute per litre of sample under optimal
conditions and at defined temperature . It is expressed as IU/L

• Katal – catalytic unit – One Katal is defined as the number of

mole of substrate transformed per second per litre of sample. It
is abbreviated as kat or k

6
ENZYMES IN MEDICINE
Transferases
• Diagnostic indicators – the activities of many enzymes are
routinely determined in plasma ( rarely in tissue biopsies) for
diagnostic purposes in diseases of the heart, liver, skeletal muscle,
pancreas and other tissues - enzyme diagnostics

• Therapeutic agents – several enzymes are used as drugs; new

approach - enzymotherapy

• Diagnostic tools – use as chemicals in clinical laboratory assays

 Specificity of Enzymes

In general, there are four distinct types of specificity:

Absolute specificity - the enzyme will catalyze only one
reaction.
Group specificity - the enzyme will act only on molecules
that have specific functional groups, such as amino,
phosphate and methyl groups.
Linkage specificity - the enzyme will act on a particular type
of chemical bond regardless of the rest of the molecular
structure.
Stereochemical specificity - the enzyme will act on a
particular steric or optical isomer.
 Specificity of Enzymes
One of the properties of enzymes that makes them so important
as diagnostic and research tools is the specificity they exhibit
relative to the reactions they catalyze.
Greater specificity is achieved in three ways:
1. Interpreting investigations in the light of clinical
features
2. Test pattern recognition
3. Isoenzyme determination:
AST may be due to MI or Hepatitis so, it makes confusion
in diagnosis to be confirmed by LDH levels.
ALP in Cholestasis & bone diseases :
Differentiated by bilirubin & transaminase levels in Cholestasis
Confirmed by GGT in Cholestasis.
Diagnosis of various diseases

Plasma
enzymes Isoenzymes
 ENZYMES OF CLINICAL INTEREST
Acid phosphatase (ACP)
Amylase (AMS)
Alanine aminotransferase (ALT)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (AST)
Creatine kinase (CK)
Gamma-glutamyltransferase (GGT)
Glucose 6-phosphate dehydrogenase (G6PD)
Lactate dehydrogenase (LDH or LD)
Lipase (LPS)
Plasma cholinesterase
 ENZYMES IN CLINICAL DIAGNOSIS
secretory - produced by tissues (namely liver), acting in plasma –
prothrombin, plasminogen, cerruloplasmin, choline
esterase; lipoprotein lipase
Enzymes

intracellular – function intracellulary, have no physiological use in

plasma
- membrane bound – ALP, GMT
- cytosolic – ALT, AST, LD, MDH
- mitochondrial – AST, GMDH
- lysosomal - ACP

- tissue specific – glucose-6-phosphatase – liver

amylase – pancrease
LD1 – heart
• Healthy individuals - levels of intracellular enzymes fairly constant, low –
the rate of enzyme release from damaged cells into plasma balanced by the
rate of removal of enzyme protein from plasma

Physiological enzyme levels  reference values of the enzyme activities

(determined in clinical laboratory – each lab

has its own reference values)

• Elevated enzyme activity in the plasma – reflect tissue damage accompanied

by increased release of intracellular enzyme
Skeletal muscle during exertion – physiologically elevated levels of muscle
enzymes in plasma

• Many diagnostically important enzymes = isoenzymes – pattern of

isoenzymes in plasma (determined electroforetically)
– a means of identifying the damaged tissue
ALTERATION OF ENZYME PLASMA LEVELS

Increased values – increased cell membrane permeability

anoxia, disturbances of energy metabolism 

cytosolic enzymes – ALT, LD, CK
- cell necrosis  membrane-bound enzymes – ALP, GMT
mitochondrial enzymes – AST, GMDH
- induction of the enzyme synthesis  drugs – ALP, GMT

Decreased values – inhibition of the activity  drugs

- inhibition of the synthesis  cell damage, drugs
 Examples of enzymes commonly assayed for diagnostic purposes

Enzyme Location Cause of elevated plasma level

Acid phosphatase - ACP Prostate Prostatic cancer

Alkaline phosphatase – ALP Bone, liver Rickets, hypoparathyroidism,

osteomalacia, obstructive
jaundice, cancer of bone/liver

Alanine aminotransferase – ALT Liver (muscle, Hepatitis, jaundice, circulatory

heart, kidney) faillure with liver congestion

Aspartate aminotransferase – AST Heart, muscle, Myocardial infarction, muscle

red cells, liver damage, anemia, hepatitis,
circulatory faillure with liver
congestion

Amylase - AM Pancres Acute pancreatitis, peptic ulcer

-Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver

pancreas damage, cholestasis
 Isoenzymes
Study of isoenzyme increases the
specificity of non functional plasma
enzymes for various tissue

They are different molecular forms of the same

enzyme synthesized by different tissues
 Isoenzymes
• Catalyze the same
reaction

Same • Act on the same

substrate
• Use the same
coenzymes

• Subunits
• Vmax and Km
• Physical properties
Different • Electrophoretic
mobility,
• Immunogenic
properties
• Heat stability
 Isoenzymes
Catalyse same reactions but are formed from structurally
different polypeptides.
They perform the same catalytic function.
Different isoenzymes may arise from different tissues and
their specific detection may give clues to the site of
pathology.
Various isoenzymes of an enzyme can differ in three major
ways:
- enzymatic properties
- physical properties (e.g heat stability)
- biochemical properties such as amino acid composition
and immunological reactivity's.
 Enzyme activity
Enzyme assays usually depend on the
measurement the catalytic activity of the enzyme,
rather than the concentration of the enzyme
protein itself.
 Examples of clinically important isoenzymes:
1-Lactate dehydrogenase
2-Creatinekinase.
3-Alkaline phosphatase
Lactate dehydrogenase has Five isoenzymes present in blood
LDH1(HHHH) specific for HEART

LDH2(HHHM) " " RBCs

LDH3(HHMM) " " BRAIN

LDH4(HMMM) " " LIVER

LDH5(MMMM) " " MUSCLE

 Lactate dehydrogenase (LDH or LD)
Converts pyruvate to lactate (and vice versa) during and after
anaerobic metabolism.
LDH occurs as a tetramer of 2 different subunits:
LD-1 (HHHH) from the heart: Elevated after MI.
LD-2 (HHHM) from the kidney: Elevated after renal
infarction.
LD-3 (HHMM) from the lung, spleen and pancreas: Elevated
in pulmonary embolism.
LD-4 (HMMM) and LD-5 (MMMM), both from the liver and
skeletal muscle: Elevated in injury to liver or skeletal muscle.
LDH Isoenzymes

Control

1 2 3 4 5
 Electrophoresis of LDH

In myocardial infarction :Total serum LDH level increases

due to elevation of LDH1 (cardiac) isozyme, electrophoresis
is important to detect which fraction increased
Creatinekinase(CK)
Three isoforms are present
-CK 1 (BB) (Brain)
-CK2 (MB) (Heart)
-CK3 (MM) (Skeletal muscle)

In myocardial infarction there

is elevation of total CK with
marked elevation of CK2 (MB) after 4-8
hours
electrophoresis is important to detect
which fraction increased
Examples of isoenzymes commonly assayed for diagnostic purposes

Enzyme Location Cause of elevated plasma level

Creatine kinase – CK
CK-MB Heart Myocardial infarction
CK-MM Skeletal muscle Muscular dystrophy

Lactate dehydrogenase – LD
LD1 > LD2 Heart, kidney, Myocardial infarction, kidney
blood cells disease, megaloblastic anemia,
leukemia
LD2, LD3 Leukemia
LD5 Liver, muscle Liver disease, muscle damage
Alkaline phosphatase
ALP isoenzymes, present in blood, are derived from bone,
liver, intestine, or placenta of pregnant woman
 Information from enzymes measurements

Presence of disease
Organs involved
Aetiology /nature of disease: differential diagnosis
Extent of disease-more damaged cells-more leaked
enzymes in blood
Time course of disease
ENZYME-ORGAN ASSOCIATIONS

ORGAN ENZYME
Liver Aminotransferases (AST, ALT)
Lactate DH (LD5)
Gamma-glutamyltransferase
Alkaline phosphatase
Heart Creatine kinase (MB)
Lactate DH (LD1 > LD2)
Troponins I and T
Skeletal muscle Creatine kinase (MM)
Lactate DH (LD5)
Aldolase
ENZYME-ORGAN ASSOCIATIONS

Brain Creatine kinase (BB)

Bone Alkaline phophatase (heat labile)

Prostate Acid phosphatase

Pancreas Amylase
Lipase
 HOME TAKE MESSAGE
 Enzymes are biological catalysts present in every cell of the body.
 An enzyme will act on a specific substrate yielding a product.
 An isoenzyme is a genetic variant produced largely within a specific tissue.
 Isoenzyme patterns can give information about organ-specific disease.
 Important enzymes in the investigation of heart disease are CK, LDH and AST.
 Important enzymes in the investigation of liver disease are AST, ALT, alkaline
phosphatase and GGT.
 Creatine kinase has three isoenzymes: CK-MM, CK-MB and CK-BB.
 LDH has five isoenzymes.
 Alkaline phosphatase can be used in the investigation of liver and bone disease.
 Increased levels of acid phosphatase are found in prostate cancer.
 GGT is induced by alcohol and is useful in monitoring alcohol abuse.
 Enzyme measurements should be performed using zero order kinetics, i.e. using excess
substrate.
 Determinations of enzyme activity can be performed using an end-point or kinetic
method