Biochemistry Page |1

Biological Importance of Enzymes

 Special Diagnosis Tests using Enzymes
 Enzymes Uses in Diagnosis
 Diseases caused by Deficiency of Enzymes
 Diseases Caused By excessive Enzymes Activities
 Enzymes Activators and Inducers as Medicines
 Enzymes uses as Medicines
 Enzymes Inhibitors as Medicines

Special Diagnosis Tests using Enzymes

Several sophisticated diagnostic tests have been devised in which enzymes are used as special
tools
• Polymerase chain reaction(PCR)
• Diagnosis of genetic diseases before birth
• Enzyme linked immuno sorbent assay (ELISA)
Polymerase chain reaction (PCR)
• This technique uses DNA polymerase along with its primers to obtain large amounts
of the two strands of DNA from such a small amount of DNA that can not be
analyzed. This technique has made it possible to detect the source of DNA starting
from single cell, hair follicle or spermatozoon.
Diagnosis of genetic diseases before birth
• This is done by the study of the so called restriction fragment length polymorphism
(RFLPs). The enzyme restriction endonuclease is used to cleave both strands of DNA,
depending upon the base sequence at highly specific sites to produce RFLPs.
• The RFLPs so obtained are compared to the normal ones to find out any differences
which help in diagnosing diseases such as sickle cell disease, Huntington’s chorea etc.
Enzyme linked immunosorbent assay ELISA
• Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of
antibodies with the sensitivity of simple enzyme assays, by using antibodies or
antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful
measurement of antigen or antibody concentration.

Biological Importance of Enzymes

 Biochemistry Page |2

Enzymes Uses in Diagnosis

Amylase
• It is increased in Parotitis and acute Pancreatitis.
Cholinesterase
• It is decreased in Liver disease and raised in Nephrotic syndrome.
Alkaline Phosphatase
• It is raised in rickets, hyperparathyroidism, paget’s disease, osteoblastic sarcoma and
obstructive jaundice.
Acid Phosphatase
• It is increased in prostate carcinoma with metastases; this is due to its greater
production.
Serum glutamate oxaloacetate transaminase (SGOT) also called serum aspartate
aminotransferase (Serum AST)
• It is raised in acute myocardial infarction.
• It is also raised in acute liver damage(hepatitis)
Serum glutamate pyruvate transaminase (SGPT) also called alanine aminotransferase
(serum ALT)
• It is raised in acute liver damage.
• On injury to liver cells in which it is present in large amount, this enzyme leaks into
blood stream
Lactate dehydrogenase (LDH)
• It is raised in myocardial infarction; it is also raised in acute liver damage. Its
isozymes measured which gives more specific information.
Creatine kinase(CK) (Creatine phosphokinase CPK)
• It is raised in diseases of the muscle and in myocardial infarction. Its isozymes
measurement diagnosis specific.
Aldolase
• It is raised in pseudo hypertrophic muscular dystrophy.
Gama glutamyl Trans peptidase (GGT)
• Its level is raised in hepatobiliary obstruction and alcohol induced liver disease.
Creatine phosphokinase (CPK)
• It (CKMB) is raised in acute myocardial infarction and Cktotal in skeletal muscle
disorder

Biological Importance of Enzymes

 Biochemistry Page |3

Disease caused by deficiency of enzymes

A large number of diseases result from deficient or defective enzymes. Some examples of
diseases are:
i) Phenylketonuria
• It is caused by the deficiency of Phenylalanine hydroxylase
ii) Albinism
• It is caused by the deficiency of tyrosinase
• The skin and hair lack the melanin pigment
iii) Tyrosinemia
• This defect is of three type 1,2 and 3 and due to the deficiency of fumaryl
acetoacetatehydrolase, tyrosine aminotransferase and p-hydroxyphenylpyruvate
dioxygenase respectively.
iv) Maple syrup urine disease
• This is due to the deficiency of branched chain a-keto acid dehydrogenase complex
v) Glycogen storage diseases
• These are of many types and are caused by the hereditary deficiency of enzymes
taking part in glucose and glycogen metabolism

Diseases caused by excessive enzyme activity

The activities of certain enzymes may be raised either as a primary defect or an enzyme
may become overactive due to a certain factor.
Phosphoribosyl pyrophosphate [PRPP] synthase
• Over activity of this enzyme causes overproduction of uric acid resulting in gouty
arthritis and nephrolithiasis
Pyrimidine 5’-nucleotidase
• Its over activity causes developmental delay, seizures, ataxia and language defect
• δ ALA Synthase
• Its over activity cause porphyria

Enzyme activators & Inducers as Medicines

• In certain cases drugs are used to activate or induce a certain enzyme action.
• An important example is the use of phenobarbitone in decreasing the raised
unconjugated bilirubin level in patients of Gilbert’s syndrome and moderate variety
type-II of Crigler- Najjar syndrome

Biological Importance of Enzymes

 Biochemistry Page |4

Enzymes uses as Medicines

Many enzymes have proved very useful as drugs in those cases in whom they can be
brought directly to the site where they shall act.
E.g: To the body surface, to body cavities accessible from the outside, into the blood
stream and into the gastrointestinal tract. Some uses of enzymes are:
1) Enzyme substitution in digestive disturbances
2) Blood coagulation
3) To increase blood supply to tissue
4) Enzymes for thrombolysis
5) Enzymes for cancer treatment
6) Locally applied enzyme preparations
1) Enzyme substitution in digestive disturbances
• The enzyme preparations used for the digestive disturbances contain proteinases,
lipases and amylases of animal, vegetable and microbial origin. Pepsin and pancreatin
(containing enzymes of pancreatic juice) along with dry bile powder are the most
frequently used enzyme preparations in digestive disorders.
2) Blood coagulation
• Thrombin is used locally to stop bleeding.
3) To increase blood supply to tissue
• The enzyme kallekrein increases the formation of kinins from their precursors, i.e.
kininogens; kinins produce vasodilation and for this reason Kallikrein is used in cases
of impairment of blood supply to tissues, e.g. of the extremities and the heart.
4) Enzymes for thrombolysis
• Streptokinase originally obtained from streptococci is not an enzyme in itself, but
after combining with circulation plasminogen it comes to possess protease activity
that catalyzes the conversion of additional plasminogen to plasmin which causes lysis
of fibrin clots. Urokinase isolated from urine activates the fibrinolysis system by
directly converting plasminogen to plasmin. The enzymatic tissue plasminogen
activator is also used for this purpose.
5) Enzymes for cancer treatment
• L-asparaginase has proved very useful in the therapy of lymphoblastic leukaemia
especially in children. The amino acid asparagine, the substrate of this enzyme, is
essential for the proliferation of cancer cells. Administration of L-asparaginase
hydrolyzes asparagine and result in a decreased availability of asparagine to cancer
cells and therefore their abnormally fast growth is arrested resulting in a remission.

Biological Importance of Enzymes

 Biochemistry Page |5

6) Locally applied enzyme preparations

a) Hyaluronidase
b) Trypsin and chymotrypsin
c) RNase and DNase
a) Hyaluronidase:
This enzyme hydrolyses mucopolysaccharides such as hyaluronic acid and chondroitin
sulphate and thus causes a loosening of the deep layers of the skin. It has been used for
increasing the rate of fluid absorption from subcutaneous tissues and in the treatment of
sprains, hematomas and thrombophlebitis, etc.
b) Trypsin and chymotrypsin:
These are used to degrade necrotic tissue, mass pus, secretions and effusion thus
producing a cleaning of the wounds healings. Chymotrypsin is also used orally and by
injection in these condition
c) RNase and DNase :
These are used in the form of streptodornase for the disintegration of Pus

Enzymes Inhibitors as Medicines

These are followings:

Drug Name Name of Enzyme Action Used in

Inhibited treatment of
Diseases

Captopril Angiotensin Decreases formation of Hypertension

Converting enzyme Angiotensin II and aldosterone
(ACE)

Physostigmine Acetylcholinesterase Prolongs the action of Myasthenia

acetylcholine gravis

Acetylsalicylic acid COX-I and COX-II Inhibit Pyretic and inflammatory Fevers,
and other NSAIDs action Inflammation,
e.g. arthritis

Azidotthymidine Reverse transcriptase Suppress HIV Replication AIDS

Cardiac glycosides Na+/K+ ATPase in Increases the force of myocardial Heart failure
heart muscle contraction

Biological Importance of Enzymes

 Biochemistry Page |6

Catalytic Mechanism of Chymotrypsin

• Chymotrypsin
– Protease: catalyze hydrolysis of proteins in small intestine
– Specificity: Peptide bond on carboxyl side of aromatic side chains (Y, W, F)
& Large hydrophobic residues (Met,…)
– Three polypeptide chains cross-linked to each other
– Three catalytic residues: Ser195, His57, & Asp102

Summary for the Catalytic Mechanism of Chymotrypsin

Mechanism
General acid-base catalysis & Covalent catalysis
Two steps:
Acylation and Deacylation (rate limiting; reverse of acylation with water substituting the
amine component)
Key features
Active Ser195 & roles of the three catalytic residues
Tetrahedral transition state
Oxyanion and Oxyanion hole
Acyl-enzyme intermediate

Biological Importance of Enzymes

 Biochemistry Page |7

Catalytic Mechanism of Chymotrypsin

Biological Importance of Enzymes

 Biochemistry Page |8

Mechanism of ribonuclease

RNA structure

Biological Importance of Enzymes

 Biochemistry Page |9

Structure of RNase protein

• The size of RNase A is small
• Has 124 amino acid residues
• Contains 19 of the 20 natural amino acids,
lacking only tryptophan
• Has similar shape to a kidney with active site
residues lying in the cleft
• Long four-stranded antiparallel β-sheet and three
short α-helixes
• Cross-linked by four disulfide bonds involving
all eight of its cysteine residues

Example of acid-base catalysis

• Ribonucleases are enzymes that hydrolyze RNA by cutting the P-O ester bond
attached to ribose 5’ carbon.
• It represents a typical example of acid base catalysis.
Ribonuclease
NH2
N
N
Adenosine

O N N
O P O CH2
O
O

O
OH
O
O P O N
Ribonuclease A Uridine
O
O N
CH2
O

O OH

O P O

Biological Importance of Enzymes

 Biochemistry P a g e | 10

RNA Binding

SUBSTRATE SPECIFICITY:
• RNase A catalyzes the cleavage of the P-O bond of an RNA strand and the hydrolysis
of the P-O bond of a nucleoside 2’,3’-cyclic phosphodiester on the 3’-side of a
pyrimidine residue.
• Pancreatic RNase is specific for a pyrimidine base (uracil or cytosine) on the 3’ side
of phosphate bond that is cleaved.
• The hydrolysis of RNA catalyzed by RNase A occurs in two steps with a 2’,3’ cyclic
phosphate diester intermediate.

_____________________________________________________________________

___

Biological Importance of Enzymes

 Biochemistry P a g e | 11

Mechanism of RNase A

In the formation of 2’,3’ cyclic phosphate diester intermediate

• His 12 acts as general base and remove the proton from 2‘-hydroxy group of ribose.
• This will increase the nucleophilic character of the oxygen atom.
• His 119 will act as general acid and protonate one oxygen of the phosphate negative
charge.
• The side chain of Lys41 and the main chain of Phe120 enhance catalysis by
stabilizing this transition state

Biological Importance of Enzymes