Agnes L.

Castillo, PhD
Faculty of Pharmacy
University of Santo Tomas
Enzymes
they are high molecular weight proteins which
regulate the biochemical processes in the
organism
Exerts a catalytic effect on chemical reactions, or
they are special organic catalyst.
Biological catalyst a substance that increases
the rate, or velocity of a chemical reaction
without itself being changed in the overall
process.
Enzymes
are special proteins produced only by living cells
which in combination with various non-protein
factors
promote and control with a high degree of
specificity the chemical processes of life.
Their molecular weights vary from about 9000
(hydrogenase) to about 1,000,000.
Characteristics of Enzymes

1. All enzymes are protein in nature,

with the general chemical and
physical properties of such
compounds, including instability to
heat, radiation, etc.
Characteristics of Enzymes
2. Many enzymes will operate with
assistance of a non-protein co-factor.

CO-FACTOR water-soluble, dialyzable

chemical factor which may be
:
a) simple inorganic or metallic ion =
activator
b) organic cofactor = co-enzyme
 Cofactors
When tightly bound =
Non-protein
component PROSTHETIC GROUP

APOENZYME HOLOENZYME
+ COFACTOR (active)
(inactive)

Organic molecule Metal ion

(COENZYME) Fe+2/Fe+3
Biotin Cu+2
Co-A Zn+2
FAD Mg+2
NAD+;NADP+ Mn+2
TPP K+
PP Na+
Lipoic acid
Cobamide
coenzyme
THF
Features of Enzymatic Catalysis
Rates proceed under physiological conditions
Specificity
Catalytic activities highly regulated
Localization within the cell
3. Enzymes operate at a very high
speed.
Most enzymatic reactions, under optimal
conditions, proceed 108 to 1011times
more rapidly than the corresponding
nonenzymatic reactions.
 4. Enzyme reactions are frequently reversible.

The direction in which a reaction proceeds is

determined by the condition of the cells.

Lactic acid+NAD LDH pyruvic acid+ NADH

5. Some enzymes are synthesized in
an inactive state. These proenzymes
or zymogens become active only
when converted by specific agents,
such as hydrogen ions.

HCl
Pepsinogen Pepsin

Trypsinogen enterokinase Trypsin

 Pepsin is a proteolytic enzyme found in
the gastric juice.
It is most active at a pH of about 1.8,
but in neutral or alkaline media, pepsin is
entirely inactive.
It converts proteins into proteoses and
peptones.
 Trypsin is a proteolytic enzyme ,
more active than pepsin

converts proteoses and peptones

into polypeptides & amino acids.

It acts best in an alkaline medium of

about pH 8.
 6.It is usual to describe enzymes as being
extremely specific
they confine their activity to one particular
substrate.

Absolute specificity catalyze a single unique

reaction and act on a particular substrate.
Ex.Sucrase, maltase, urease, cellulase etc.
Group specificity an enzyme acts on
different substrates falling under a
particular group.

Ex.Lipase,phospholipase,acetycho
linesterase = ex. Of esterases
Stereospecificity- the enzyme in this
case attacks one member of a pair of
stereoisomers which differs only in the
arrangement of atoms, and are
otherwise identical.

Ex. Glucose and galactose on

fermentation
D arginine and L- arginine using
arginase
7. The rate of enzyme reaction is greatly
influenced by a number of factors:

a) Temperature an increase in
temperature will speed up enzyme
activity.
Act best at temperatures between 35o & 40oC;
temperatures above 65oC, usually completely
destroy them, whereas their activity is negligible
at 0oC.
 Temperature
b) pH there is an optimum pH for
every enzyme.
 Enzyme concentration

pH
c) Substrate concentration as the
concentration of substrate molecules
rises, more and more of the active sites
on the enzyme molecules become
occupied.
Once all these sites are busy, the
enzyme is working at maximal capacity,
promote greater production.
d) Enzyme concentration
generally speaking, the amount
of chemical change produced is
directly proportional to the
enzyme concentration.
e) Presence of activators and inhibitors.

Enzyme Inhibition loss of enzyme

activity as a result of the action of
chemical agents on the enzyme.
Two forms of enzyme inhibition:
a) Competitive inhibition inhibiting
agents binds to the enzyme at the
same site as the substrate,
substrate & inhibitor compete for
the same position on the enzyme
surface.
b) Non-competitive inhibition inhibitor
binds to the enzyme at a point other
than that at which the substrate is
bound.
The enzyme may remain active but the
bound inhibitor has so modified its
structure that the rate of activity is
diminished.
Enzyme denaturation

loss of enzyme activity as a result of a

physical process like action of heat,
mechanical agitation or irradiation.
It may be partial or total, reversible or
irreversible.
 Factors affecting rate of enzyme reaction

f) Nature and concentration of

products.

g) Time the longer the reaction

proceeds, the greater will be the
concentration of the products thereof.
8. When an enzyme is produced by
more than one type of tissue, it can be
shown by electro- phoresis and other
techniques.
A group of enzymes with identical main
functional features but differing in
physical & chemical properties is called
isoenzymes or isozymes.
Ex.
Alkaline phosphatase: bone,placenta,
liver, intestine.
Acid phosphatase: prostatic(prostate
gland) non-prostatic ( rbc, platelets,
liver)
Aldolase: liver, muscle;
Lactate dehydro genase: heart, liver,
rbc, skeletal muscle.
Creatine kinase: muscle, heart, brain.
Purification of Protein & Separation
techniques:

Electrophoresis :
to separate individual proteins from one
another
to ascertain the purity of protein
preparation
to estimate the number of components
present in a protein mixture.
This method may also be used to cause a
desire component to migrate away from an
undesired impurity.
 Denaturation
The most usual method of separation of
protein & non- protein material
From an extract containing all soluble
components of a plant tissue,
proteins may be removed by heating 70
100oC
and filtration or centrifugation of the
denatured ppt from the soluble low molecular
weight compound.
High concentration of acid may be used
& 5 15% trichloroacetic acid is effective in
precipitating most proteins even at low
temperature.
A tissue may be dried in the oven in which
case the denatured protein remain in the
residue during a subsequent water extraction.
Fractional precipitation w/ concentrations of
neutral salts, generally ammonium sulfate to
separate proteins. Other proteins are ppted only
at higher concentration.
Kohn technique- successive addition of
acetone, alcohol or deoxane to aqueous protein
solution may be used for protein separation.
- This method has been used for preparing many
enzymes & in particular the fractionation of
human blood serum proteins.
 Ultracentrifugation
the molecules of proteins in this case are
actually sedimented from solution by application
of very gravitational fields. Since heavier
molecules sediment more rapidly than lighter
molecules.
Other methods of protein separation include:
- absorption of proteins in suitable
absorbents
- partition chromatography on starch or
filter paper.
Enzyme Nomenclature:
1. Name of substrate or group on which the
enzyme acts + suffix ase .
2.Type of reaction involved.
Ex. Oxidases catalyzing oxidation process.
3. Combination of # 1 and # 2.
4. Trivial or empirical names which give no
clues for the enzymes chemical structure. Ex.
Trypsin, steapsin, pepsin, papain etc
Nomenclature & Classification
 Units of Enzyme Activity
Enzymes are measured by activity
rather than concentration.
Activity is expressed based on:
1] increase in concentration of one of
the products
2] decrease in concentration of
substrate
3] rate of change in concentration of
coenzyme as a measure of rate
of reaction

may be expressed in units,

milliunits,microunits, etc per milliliter of
sample
 Units of Enzyme Activity
1 unit = amount of enzyme that catalyzes the
conversion of one mol of substrate or
coenzyme per minute under the
defined conditions of the test (optimum
pH & T and substrate concentration)
= mole/min
 Units of Enzyme Activity
1U = 1 mol/min
1mU = 1 millimicromol/min
1U = 1 micromicromol/min

katal one mole of substrate catalyzed

per second
 Proteolytic Enzymes
HCl
Pepsinogen Pepsin

Trypsinogen enterokinase Trypsin

Pepsin obtained from glandular layer of the

fresh stomach of the hog, Sus scrofa var.
domesticus (Suidae)
 Proteolytic Enzymes

Casein rennin curdles

Proteoses & Peptones erepsin amino

acids
Proteolytic Enzymes
Trypsin & Chymotrypsin crystallized
from an extract of the pancreas of the
ox Bos taurus (Bovidae)

Proteins pepsin proteoses trypsin peptides &

& peptones amino acids

 Proteolytic Enzymes
Pancreatin - contains amylase, lipase & protease
obtained from the pancreas of the hog, Sus scrofa var.
domesticus (Suidae) or of the ox, Bos taurus
(Bovidae)

Papain from the dried & purified latex of the fruit of

Carica papaya (Caricaceae)
- digestant for proteins
- can act in acidic, neutral and alkaline pH
uses: 1) meat tenderizer, digestant
2) relieves episiotomy (surgical incision of vulva)
3) ingredient in cleansing solutions for contact
lenses
 Proteolytic Enzymes
Chymopapain non-pyrogenic proteolytic
enzyme obtained from the latex of papaya.
treatment of herniated lumbar intervertebral
discs, relieves lower back pains

Bromelin / Bromelain a mixture of protein

digesting enzyme & milk-clotting enzymes
obtained from the juice of the pineapple plant,
Ananas comosus (Bromeliaceae)
- anti-inflammatory, increase tissue repair
(episiotomy)
 Amylolytic Enzymes
sucrase/invertase
SUCROSE glucose + fructose

maltase
MALTOSE glucose

zymase
GLUCOSE/FRUCTOSE alcohol + CO2

emulsin glucose + CHO + HCN

AMYGDALIN
myrosin
SINALBIN acrinyl isothiocyanate

myrosin
SINIGRIN allyl isothiocyanate
 Amylolytic Enzymes
Malt Extract - from barley, a dried grain of
one or more varieties of Hordeum vulgare
(Graminae)
- with digestive & nutritive
properties; serves as an aid in digesting
starch

thrombin
Fibrinogen Fibrin
Classification of Proteins:
This classification is based on their products
of hydrolysis, solubilities, precipitation by
salt & coagulation by heat.

1. SIMPLE PROTEINS: are those which

yield only (alpha) amino acids when they
are hydrolyzed.
a) ALBUMINS:
are proteins which are soluble in water and in
dilute neutral salt soln.,
precipitated by saturated ammonium sulfate and
coagulated by heat.
They occur in plants in small quantities in the
seeds and cell sap.
Plant albumins isolated are leucosin in seeds of
wheat, rye , barley;
legumelin in seeds of soy bean and cow pea;
ricin in seeds of castor bean.
 b) GLOBULIN: insoluble in water but soluble in
saline soln (5 10%), precipitated by partial
saturation with ammonium sulfate at different
degrees of saturation & is coagulated by heat.
Plant globulins that have been isolated are :
Legumin in seeds of pea & bean; tuberin in potato
tuber
Orgenin in seeds of cow pea; maysin in seeds of
corn
Glycenin in seeds of soybean; archin in seeds of
peanut
Phaseolin in seeds of kidney bean; cucurbitin in
seeds of squash
 c) GLUTELINS:
are insoluble in water, neutral solvents, salt solns.
and alcohol
soluble in dilute acids or alkalies.
They represent a small group of proteins that are
exclusively of plant origin.
Examples are glutenin in the seed of wheat ,
oryzenin in the seeds of rice & maize ,glutelin in
the seeds of corn.
Glutelins and Prolamines are collectively called
glutens
 d) PROLAMINES:
are insoluble in water and salt soln.
but are soluble in dilute acids or alkalies & in
alcohol (70 80%).
On hydrolysis they yield a relatively large amount
of proline & amide nitrogen & this is how they got
their name prolamine.
Examples are gliadin in the seeds of wheat & zein
in the seeds of corn.