ENZYMES 3.

HYDROLASE
- Catalyzes the hydrolysis of a chemical bond
- biomolecules with catalytic activity
- adds water
- biochemical reactions in the body are sustained
- can undergo epoxidation and cleavage/breaking
by enzymes
into alcohols
 they are more sufficient catalyst than
- invertase – catalyzes hydrolysis of sucrose into
inorganic catalysts
fructose and glucose
 SUBTRATE SPECIFIC
- EC 3.1 (act on ester bonds)
 REACTION SPECIFIC - functional
- EC 3.2 (act on sugars)
Factors that affect enzymes

1. Temperature
2. pH of the local environment
3. Concentration of the substrate
4. Presence of inhibitors
5. Presence of cofactors and coenzymes

TYPES

1. OXIDOREDUCTASE
4. LYASE
- Transfer of one or more electrons from a
- Catalyzes the nonhydrolytic cleavage of single
hydrogen acceptor (base) or electron donor to a
chemical bonds, leaving double bonds or a ring
hydrogen donor (acid)
structure
- Dehydrogenation (CH2OH to CH=O to COO) ,
- Decarboxlation, dehydratase
oxidases, reductases

5. ISOMERASE
- Catalyzes a spatial rearrangement of the
2. TRANSFERASE substrate molecule
- Transfers a functional group from one substrate
to another
- Nagdadagdag

6. LIGASE
- joins two molecules together
- require an energy molecule like ATP
- reaction is accompanied by hydrolysis

*enzymes named by International Union of Biochemistry

and Molecular Biology’s Enzyme Commission [EC]
numbering system
INVERTASE
- Yeast derived enzyme
- aka β-fructofuranosidase (EC3.2.1.26)
- classified as hydrolase – hydrolysis of the
terminal nonreducing β-fructofuranosidase
residues
- split sucrose into fructose and glucose
 from Saccharomyces cerevisiae (Baker’s Yeast)
and exist in 2 isoforms
 extracellular invertase
- as glycoprotein
 intracellular invertase
- as protein only
- Does not contain cysteine pH
 Boiling denatures the invertase solution
 Benzoic acid – used as a preservative in the
preparation
 Fructose *one of the inverted sugar* - sweeter
than sucrose (table sugar)
Acid and Enzymatic Hydrolysis of sucrose yields
equimolar concentration of Glucose and Fructose:
INVERT SUGAR
3,5 - Dinitrosalicylic Acid (DNS) Assay
- Used to monitor enzymatic activity
PRINCIPLE: DNS reacts with sugars to form
Aminonitrosalicylic Acid (ANS). DNS does not react
with sucrose (a non-reducing sugar) Amylase (pancreas)
IUPAC: 2-hydroxy-3,5-dinitrobenzoic acid
3-amino-5-nitrosalicylic acid
- Rate of reaction (enzyme activity) is monitored Temperature
(colorimetrically) by measuring the amount of
reaction products (reducing sugars – equimolar
mixture of Glucose and Fructose) that react with
DNS reagent
REAGENTS:
 Conc. HCL – for complete hydrolysis of
glycosidic bonds
 0.5N KOH – to neutralize excess acid
 1% DNS reagent
 DNS – oxidizing agent
 Na2SO3 – stabilizes the red color
 NaOH – increases reactivity of
sugars; changes the pH of the
reaction vessel (along with ANS
production) halting the invertase
reaction
- In alkaline solution (pH > 8), there may be partial
destruction of cysteine residues due to base
catalyzed b-elimination reactions
- In acid solution (pH <4), hydrolysis of the labile
peptide bonds, sometimes found next to
aspartic acid residues, may occur
ENZYME pH OPTIMUM
Lipase (pancreas) 8.0
Lipase (stomach) 4.0-4.5
Lipase (castor oil) 4.7
Pepsin 1.5-1.6
Trypsin 7.8-8.7
Invertase 4.5
6.7-7.0
Catalase 7.0
- Within the temperature range in which the
enzyme is stable and retains its full activity
- When temperature range is beyond 10-50OC
at its optimum, enzymes are denatured followed
by a decrease in enzyme activity
- Optimum temp. of invertase: 55oC
 The rate of an enzyme-catalyzed reaction
increases as the temp. is raised
 Enzymes will be deactivated at even moderate
temperatures
 Storage of enzymes at 5oC 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 by loss of biologic
activity)
 To minimize loss of activity on storage, even
moderate temp. should be avoided. Most
enzymes are stable for months if refrigerated at
0-4oC
 Cooling below 0oC, in the presence of additives
(e.g. glycerol) which prevents freezing, can
generally increase storage stability even further
EXP
pH & Temperature
Essential points:
- Five minutes was the reaction time across all the
temperatures
- The ice bath serves as a STOP reaction because it
will decrease substantially the effect of invertase
ENZYME INHIBITION
Inhibitor – interferes with the activity of an enzyme
- If the substance/extract, such as ACARBOSE
possesses alpha-amylase inhibitory activity, the
intensity of blue color will be more
- The intensity of blue colour in test sample is
directly proportional to alpha amylase inhibitory
activity
ALPHA AMYLASE ACTIVITY
Essential points:
- Acarbose is a complex oligosaccharide that
delays the digestion of ingested carbohydrates
= pinipigilan nya conversion ng starch
 Dose dependent on post prandial blood
sugar effect
SIDE EFFECT: diarrhea and flatulence because of
undigested carbohydrates
- 0.1 M HCL is used to STOP the reaction

- Prevent the substrate from binding to the active
site of the enzyme
- Poisons are inhibitors, as are many drugs
ALPHA AMYLASE ASSAY
PHARMACEUTICAL ENZYME INHIBITORS
Drugs act out as:
 Direct Enzyme Inhibitor
 Suppressor of Gene Function

- In humans, digestion of starch involves several ENZYME DRUG

stages β-lactamase
Antibiotic Penicillin
 Partial digestion inhibitor
- Salivary amylase – degradation of polymeric Captopril,
Cardiac ACE – inhibitors
substrates into shorter oligomers Enalapril
- Pancreatic amylase into maltose, maltotriose Antidepressant MAO – Inhibitors Selegiline
and small malto-oligosaccharides Carbonic
Diuretic Acetazolamide
- Inhibition of alpha amylase can lead to reduction Anhydrase Inhibitor
in post prandial (after eating) hyperglycemia in
diabetic condition Antimetabolites:
- Chemotherapy consists this strategy
PRINCIPLE: - A drug (mimic)to the normal metabolite is called
• Hydrolysis of starch in presence of alpha-amylase an antimetabolite
enzyme. - “fooling” an enzyme and producing a counterfeit
• Quantified by using iodine metabolite which inhibits another enzyme
 Cannot be utilized by the cell for growth
- Gives blue color with starch = iodo starch or reproduction
complex
- Reduced intensity of blue colour indicates the *look at PHARMACEUTICALLY IMPORTANT ENZYMES pic*
enzyme-induced hydrolysis of starch in to *look at ntbk*!!!
monosaccharides