ENZYMES 3.

Hydrolase: Catalyzes the hydrolysis of a chemical

- Biomolecules with catalytic activity bond. Uses water
- Biochemical reactions in the body are sustained by
enzymes
- More sufficient catalyst than inorganic catalyst
- Substrate specific
- Reaction specific

FACTORS THAT AFFECTS ENZYMES 4. Lyase: Catalyzes the nonhydrolytic cleavage of single
1. Temperature chemical bonds, leaving double bonds or a ring
2. pH of the local environment structure.
3. Concentration of the substrate
4. Presence of inhibitors
5. Presence of cofactors and coenzymes

5. Isomerase: catalyzes a spatial rearrangement of the

substrate molecule.
ENZYMES
1. Oxidoreductases
2. Transferases
3. Hydrolases - EC 3.2.1.26
Category: EC 3.1 (act on ester bonds)
Category: EC 3.2 (act on sugars – glycosylases) 6. Ligase: joins two molecules together; requires an
i. The first number shows to which of the six main energy molecule like ATP; reaction is accomplished by
divisions (classes) the enzymes belongs hydrolysis
ii. The second figure indicates the subclass
iii. The third figure gives the sub-subclass Invertase
iv. The fourth figure is the serial number of the • Yeast derived enzyme
enzyme in its sub-subclass • Official name: ß-fructofuranosidase (EC 3.2.1.26)
4. Lyases • Classified as hydrolase – catalyzing hydrolysis of the
5. Isomerases terminal nonreducing ß-fructofuranosidase residues
6. Ligases • Splits sucrose to glucose and fructose
*named by International Union of Biochemistry and Molecular
Biology’s Enzyme Commission[EC] numbering system

TYPES OF ENZYMES
1. Oxidoreductase: transfer of one or more electrons
from a hydrogen acceptor or electron donor to a
hydrogen donor • From Saccharomyces cerevisiae (Baker’s Yeast) and
exists in 2 isoforms
o Extracellular invertase – as glycoprotein
o Intracellular invertase – as protein only, does
not contain cysteine
2. Transferase: transfers a functional group from one • Boiling denatures the invertase solution
substrate to another. * Invert Sugar
• Benzoic acid – used as preservative in the preparation
of invert sugar
• *fructose – sweeter than sucrose (table sugar)
• Acid hydrolysis and enzymatic hydrolysis of sucrose
yields equimolar concentration of glucose and
fructose: Invert sugar
Dinitrosalicylic Acid (DNS) Assay
• Dinitrosalicylic acid (DNS) Assay
o This method is used to monitor enzyme
activity.
• Principle involved:
o DNS (3,5-dinitrosalicylic acid, IUPAC name 2-
hydroxy-3,5-dinitrobenzoic acid) reacts with
reducing sugars (eg. glucose and fructose) to
form 3-amino-5-nitrosalicylic acid (ANS). DNS
does not react with sucrose (non-reducing
sugar).
• pH dependence of enzyme activity is a consequence
of acid-base behavior or changing degree of ionization
of groups in the enzyme. In the substrate, or in both.
ü The changes in charges with pH affect the activity,
structural stability and solubility of the enzyme.
• Extremely high or low pH values generally result in
complete loss of activity for most enzymes
• Invertase exhibits high activity over a broad pH range
3.5-5.5
• With optimum pH near 4.5

● In alkaline solution (ph > 8), there may be partial

destruction of cystine residues to base catalysed b-
elimination reactions
● In acid solutions (pH < 4), hydrolysis of the labile
• Rate of reaction (enzyme activity) is monitored peptide bonds, sometimes found next to aspartic acid
(colorimetrically) by measuring the amount of reaction residues, may occur
products (reducing sugars equimolar mixture of Enzyme pH Optimum
glucose and fructose) that react with DNS reagent. Lipase (pancreas) 8.0
Lipase (stomach) 4.0 - 5.0
Lipase (castor oil) 4.7
Pepsin 1.5 - 1.6
Trypsin 7.8 - 8.7
Invertase 4.5
Amylase (pancreas) 6.7 - 7.0
Catalase 7.0

Temperature

Reagents for DNS assay

• Conc HCl – for complete hydrolysis of glycosidic
bonds
• 0.5N KOH – to neutralize excess acid
• 1% DNS reagent
o Dinitrosalicylic acid – oxidizing agent
o Na2SO3 – stabilized the red color
o NaOH – increases the reactivity of sugars;
changes the pH of the reaction vessel (along
with ANS production) halting the invertase
reaction.
Factors Affecting Enzyme Activity
pH
● Rate of reaction increases with temp; within the temp
range in which the enzyme is stable and retains its full
activity. When the temp range is beyond 10-50° at
optimum, enzymes are denatured followed by a
decrease in enzyme activity
● Optimum temp of invertase = 55°C
● The rate of an enzyme-catalyzed reaction increases as
the temperature is raised
● Enzymes will be deactivated at even moderate
temperatures. Storage of enzymes at 5°C or below is
generally the most suitable. Some enzymes lose their
activity when frozen
● Enzymes, however, are proteins and undergo
essentially irreversible denaturation (i.e.
conformational alteration entailing a loss of biological
activity) at temperatures above those to which they are
ordinarily exposed in their natural environment
 ● In order to minimize loss of activity on storage, even
moderate temperatures should be avoided. Most
enzymes are stable for months if refrigerated (0-4°C).
Cooling below 0°C, in the presence of additives (e.g.,
glycerol) which prevent freezing, can generally
increase this storage stability even further

pH and Temperature
Essential points:
● Five minutes was the reaction time across all the
parameters
● The ice bath serves as a STOP reaction because it will
decrease substantially the effect of invertase

ENZYME INHIBITION
An inhibitor is a molecule that interferes with the activity of an
enzyme.
- Prevent the substrate from binding to the active site of
the enzyme
- Poisons are inhibitors, as are many drugs.

ALPHA AMYLASE ASSAY

In humans, the digestion of starch involves several stages.
- Partial digestion
• Salivary amylase - degradation of polymeric substrate
into shorter oligomers.
• Pancreatic amylase into maltose, maltotriose and small ALPHA AMYLASE ACTIVITY
malto-oligosaccharides. • Acarbose is a complex oligosaccharide that delays the
- Inhibition of alpha-amylase can lead to reduction in post digestion of ingested carbohydrates
prandial hyperglycemia in diabetic condition. o Dose dependent post prandial blood sugar
effect
PRINCIPLE • COMMON SIDE EFFECT: diarrhea and flatulence (gas)
• Hydrolysis of starch in presence of alpha-amylase because of undigested carbohydrates
enzyme. • 0.1 M HCl is use to STOP THE REACTION
• Quantified by using iodine
ü Gives blue color with starch = iodostarch complex PHARMACEUTICAL ENZYME INHIBITORS
Drugs acts out as
ü Reduced intensity of blue colour indicates the
• Direct Enzyme Inhibitor
enzyme-induced hydrolysis of starch in to
• Suppressor of Gene Function
monosaccharides
ENZYME DRUG
ü If the substance/extract, such as ACARBOSE
Antibiotic B-Lactamase Penicillin
possesses alpha-amylase inhibitory activity, the
Inhibitor
intensity of blue color will be more
Cardiac ACE - Inhibitors Captopril
ü The intensity of blue colour in test sample is Enalapril
directly proportional to alpha amylase inhibitory Antidepressant MAO - Inhibitors Selegiline
activity Diuretic Carbonic Acetazolamide
Anhydrase
Inhibitor
Antimetabolites:
• Strategy of chemotherapy consists
• A drug (mimic) to the normal metabolite is called an
antimetabolite
• “fooling” an enzyme and producing a counterfeit
metabolite.
• The counterfeit metabolite inhibits another enzyme
- Cannot be utilized by the cell for growth or
reproduction.
Name (under USP) Availability Category
Pancreatin Capsule, Tablet Digestive Aid
Trypsin Aerosol Proteolytic
(crystallized) enzyme
Chymotrypsin Opthalmic Proteolytic
solution enzyme to zonule
lysis; for cataract
surgery
Hyaluronidase Injection Adjuvant for
increase drug
absorption,
diffuse local
anesthetic
Pancrelipase Capsule, tablet Digestive aid for
Steatorrhea
Streptokinase Injection Fibrinolytic agent
for MI
Urokinase Injection Fibrinolytic agent
for MI
Alteplase Injection Fibrinolytic agent
for MI
Papain Cream (with urea) Wound
debridement