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ACTIVITY NO.

20 and 21
ENZYMES AND FACTORS INFLUENCING ENZYME ACTION
ENZYMES
Comes from the Greek words en which means in, and zyme which means yeast.
An organic compound that acts as a catalyst for a biochemical reaction.
o Catalyst speeds up the rate of biochemical reaction
o Enzyme is the most efficient catalyst that can increase the rates of reaction
by 1020
GENERAL STRUCTURAL CLASSES:
1)

2)

SIMPLE ENZYME
Is an enzyme composed of protein (amino acid chains))
CONJUGATED ENZYME
It has protein and non-protein part
o Apoenzyme is a protein part of a conjugated enzyme
o Cofactor - in a non-protein part of the conjugated enzyme ; provides
additional chemically reactive functional groups besides those present in
the amino acid chain of apoenzyme.
Vitamins (COENZYME) and METAL IONS are biochemically
important cofactors
Combination of the apoenzyme and cofactor enzyme produces a
biochemically active enzyme.
Apoenzyme + cofactor = Holoenzyme

CLASSES OF ENZYMES:
1) OXIREDUCTASE
An enzyme that catalyzes an oxidation-reduction reaction.
Oxidation reaction is an oxidation that increases the number of C-O bonds and/or
decreases the number of C-H bonds
Reduction reaction is a reduction that decreases the number of C-O and/or
increases the number of C-H bonds
Example:

2) TRANSFERASES
Is an enzyme that catalyses the transfer of a functional group from one molecule to
another
TWO MAJOR TYPES
o Transaminases catalyze the transfer of an amino group from one molecule
to another.
o Kinases catalyzes the transfer of a phosphate group from adenosine
triphosphate (ATP) to give adenosine diphosphate (ADP) and a
phospholrylated product containing an additional phosphate group; major
role in the metabolic energy-production reaction.
Example:

3) HYDROLASES
An enzyme that catalyzes a hydrolysis reaction in which the addition of water to a
molecule to a bond causes the bond to break.
It is the central process of digestion.
o The carbohydrases - effects the breaking of glycosidic bonds in oligo- and
polysaccharides
o The Proteases- effect the breaking of the peptide linkages inprotein
o The Lipases effect the breaking of ester linkages in triacyglycerols
Example

5) ISOMERASES
Is an enzyme that catalyzes the isomerization (rearrangement of atoms) of a
substrate in a reaction, converting it into a molecule isomeric with itself.
Example:

6) LIGASES
Is an enzyme that catalyses the bonding together of two molecules into one with
the participation of ATP.
o ATP involves is involved as energy provider.
4) LYASES
An enzyme that catalyzes the addition of a group to a double bond or the removal of
a group to form a double bond in a manner that does not involve hydrolysis or
oxidation.
o Dehydratase removal of water from a double bond
o Hydratase effects the addition of the component of water to a double
bond
Example:
MODELS OF ENZYME ACTION
ENZYME ACTIVE SITE
o Crevicelike location in the enzyme that participates in the in the
interaction with substrate during the reaction
o It is small part of an enzymes structure that is actually involved in catalysis.
SUBSTRATE
o The reactant in an enzyme catalyzed reaction

ENZYME - SUBSTRATE COMPLEX


o Is the intermediate reaction species that is formed when a substrate binds
to the active site of an enzyme.

Reaction rate increases with increasing enzyme concentration , if the amount of


concentration rate is kept constant and the enzyme concentration is increased, the
reaction rate increases because more substrate molecules can be accommodated in
a given amount of time.

TWO MODELS OF ENZYME ACTION:


1) Lock and Key model
The active site in the enzyme has a fixed, rigid geometrical conformation.
Only substrate with a complementary geometry can be accommodated at such a
site, much as a lock accepts only certain keys.
2) Induced fit model
Allows for small changes in the shape or geometry of the active site of an enzyme to
accommodate a substrate.

ACTIVITY NO. 20
A. POTATO OXIDASE
- OXIDASE is an enzyme that causes or promote oxidation.
- Read the article on the enzymatic browning: discoloration of fruits and
vegetables.

FACTORS THAT MAY AFFECT ENZYME ACTIVITY


1) TEMPERATURE
Temperature is a measure of the kinetic energy of molecules
Higher temperature means molecules are moving faster and colliding more
frequently.
This applies to the collisions between substrate molecules and enzymes.
OPTIMUM TEMPERATURE - is the temperature at which an enzyme exhibits a
maximum activity
o Human body has 37 degree Celsius optimum temperature to carry out
reaction
o Increase in core body temperature may lead to denaturation of enzyme
2) pH
physiological pH range 7.0-7.5
enzyme pepsin function well at pH 2.0 on the other hand trypsin function well at a
pH of 8.0.
3) SUBSTRATE CONCENTRATION
Reaction rate increases with substrate concentration until full saturation occurs,
then the rate levels off.
4) ENZYME CONCENTRATION

ANSWER THE FOLLOWING QUESTIONS.


o 1) Explain the principle of browning of fruits and vegentable (potato)
once they are exposed to air. (max of 5sentences, 2points)
o 2) Explain the effect of pH and temperature on the process of oxidation
(max 5 sentences, 2 points)
o 3) why does phenol, catechol, guaiac has white coloration, pyragallol
has brown coloration.
B. POTATO PEROXIDASE
- PEROXIDASE is an enzyme found in plant, animal and bacterial cell, which
catalyzes the transfer of oxygen from organic peroxides to other substance
which is able to receive it.

C. CATALASE
- CATALASE is an enzyme found in most living cells that catalyzes the separation
of hydrogen peroxide into gaseous oxygen and water.

C. TEMPERATURE
3 testtubes with the same amount of substrate and enzyme were exposed to
different temperature. The result shows that the color changes becomes more
intense as the temperature increase, and indication that temperature affects
enzyme action, wherein increasing temperature increases the enzyme activity.
Note: that enzyme exhibit optimum activity at 37 degrees celcius, beyond cause
enzyme denaturation, below causes enzyme inactivation.

ACTIVITY NO. 21
A. SUBSTRATE CONCENTRATION
In this set up the catechol (substrate) is increasing in each of the testube with the
same concentration of enzyme, result of the experiment shows that the intensity
of color reaction becomes darker as the concentration substrate increase the rate of
enzyme action increases to its maximum.

Scanpix pp202-203
B. ENZYME CONCENTRATION
In this set up the enzyme extractl (catalyst) is increasing in each of the test tube, the
result of the experiment shows that the intensity of color reaction becomes darker.
This concludes that as the concentration enzyme increase the rate of enzyme action
increases to its maximum.

D. pH
4 test tubes with the same amount of substrate and enzyme was exposed to
different pH level, the result shows that at acidic pH the solution becomes cloudy,
increasing pH (5-light red), (7-red brown) and (9-yellow) The result indicates that
optimum activity of an enzyme is obtain at neutral pH, because low or high pH
causes the enzyme to be deactivated or denatured

ACTIVITY NO. 22 and 23


SALIVA AND PTYALIN
SALIVA
A clear , alkaline, somewhat viscous secretion produced in the mouth, containing
various salts, mucin, lysosomes, lingual lipase and salivary amylase ( responsible in
breaking down of starch)
Secreted to the amount of 1000 to 15000 cc in 24 hours. Weakly alkaline (6-7.9)
FUNCTIONS:

o Digestive ptyalin (starch digestion)


o Facilitates swallowing due to lubricating effect of mucin
o Solvent for soluble substances
o Diluents for irritant substances like alkalies ans acids
o Excretory substances like Hg, Pb and KI are eliminated through the saliva
A. SPECIFIC GRAVITY
Specific gravity of 1.007 made of 99.3% water, 0.7% solid. Organic constituent are
mucin, albumin, globulin, ptyalin, maltase, urea, cholesterol and phospholipids.
Inorganic are calcium, phosphate and carbonate.
B. pH

pH is neutral. Saliva is the first catalyst in the process of digestion. Enzyme amylase
can work efficiently at neutral pH. (exception on stomach enzyme.)
C. COMPOSITION OF SALIVA
TEST
NAME
OF
THE RESULT OBSERVED
CONCLUSION/
NO
TEST/REAGENT ADDED
PRESENCE OF
1
Molisch test
Violet ring formation
CHO
2
Biuret test
Purple color
2 peptide
3
Benedicts test
Blue
No reducing sugar
4
Dil. Acetic acid
White ppt
mucin
5
Dil. HNO3+AgNO3
White ppt
Cl6
Dil. HCL+BaCl2
White ppt
SO4
7
Dil.
HNO3+ammonium Yellow ppt
PO4
molybdate
8
Dil.
Acetic
acid+ White ppt
Ca++
(NH4)2C2)4
9
Dil.H2SO4 + KI+starch
Darck blue
NO2
10
FeCl3+Dil HCL
Yellow orange
Thiocyanate (SCN)
Post Lab Questions:
1) Same sample of saliva should be used to prevent variation of or results since the
composition varies depending on the physical state and genetic constituents of
the donor.

2) Factors responsible for the variation of the composition of saliva are as follows:
a. Mechanical factor- mastication of bland substance
b. Chemical factor action of sugars, acids and salts in the taste buds
c. Exposure to air- saliva lose CO2 and pH rises rapidly
d. Relative secretion of different glands, the release of more mucin, contain
less ptyalin.
3) Equation
a. AgNO3+Cl
AgCl ( white ppt)
b. Ba + SO4
BaSO4 (White ppt)
c. Ca + Ca2O4
CaC2O4
d. 2KI+2NO2
2KNO2+2I
e. FeCl3+3K
Fe(SCN)3+3KCL
4) Glycoprotein present in the saliva is the mucin, functions: a. Lubricant of the
mouth, B. Responsible for the viscosity of the saliva C. Protects lining of mouth
against drying.

A.

ACTIVITY NO. 23
PTYALIN
Determination of achromatic point
Iodine is the test for the presence of starch that gives dark blue color indicating a
positive result.
Benedicts reagent is a test for the presence of sugar, which give brick red ppt as a
positive result.
Beaker exposed to 40 degrees Celsius reached the achromatic point first (brown
color/no reaction) this means that the starch already dissociated and broken down.
Ptaylin an enzyme found in the saliva, having the property of converting starch into
dextrin and maltose thus aiding in the process of digestion.

B. TEMPERATURE
The test was done by exposing the ptyalin into four different temperature and the
result shows that test tube no 3 (40 degrees Celsius) reached the achromatic point
first. This indicates that ptyalin can work efficiently at optimum temperature which
is 37 degrees Celsius. Above the optimum temperature, its activity is hastened but is

rapidly inactivated due to denaturation. Below optimum temperature activity slows


down.
II. pH

Optimum pH 5.6-6.5, when the pH is lower than 4, its action is inhibited (Cabatit);
based on results of pH, below or above the optimum pH, activity ceases.
III. PRESENCE OF OTHER SUBSTANCES

-lodide and nitrates- less effective


-Sulfate, Acetates anci Phosphates- least effective
ACTIVITY NO 24
GASTRIC ANALYSIS
Importance:
1) To gain information regarding the secretory and motor activities of the stomach
2) Reveal the presence of substances which are indicative of pathological states
3) Differential diagnosis of gastric ulces from duodenal ulcer
4) Measure amount of acid produced by patient
GASTRIC CONTENT:
Gastric- having to do with or near the stomach
Function of the stomach:
Mixes saliva, food and gastric juices to form chime
Serve as a reservoir for food before release into small intestine.
Secretes juices, which contains HCl (kills bacteria and denature proteins) pepsin (begins the
digestion of protein), intrinsic factor (aids absorption of vitamins B12) and gastric lipases (aids
digestions of triglycerides)
Secret gastrin into blood.
B.Test for free HCL
1) Boas test- red streaks
2) Gunzberg Test red spots
C. Test For lactic Acid
1) Strauss Test 2 layers; ether (green); water (yellow)
2) Uffelmans test yellow brown / light brown color
D. Test for occult blood
- Blue green color
ANSWERS TO QUESTIONS:

1. Normal acidity of stomach contents is 1.6 -1.8 Stomach- columnar epithelium tining with
Halogens increases enzymatic activity but not in combination with heavy metals and
organic compounds
Chlorides, bromides and nitrates increases the activity.
ANSWERS TO QUESTIONS:

gastric pits containing gastric glands that produce gastric juice. Pepsin present digests

proteins. HCI kills bacteria

in food. lt doesn't digest food but breaksdown connective

tissues of meat.

>

lt is protected by a thick layer of mucus. lf HCI penetiates, it breakdowns/destroys the


cells.. ulcer results. Ulcer is an open sore in stomach wall caused by gradual disintegration
of tissue. lt is due to Helicobacter pyloi which impairs the ability of epithelial cells to produce

Answers to Questions:

protective mucus.

1. Ptyalin is a mixture of alpha and beta amylase (alpha dominarrt)


2. Acts upon starch and glycogen ( alpha 1-4 glycosidic bonds)
3. Op. pH. 5.6- 6.5
Op. Temp. 37 deg C.
Activators (Anions)
Chlorides and bromides -most effective

2. Lactic acid is secreted by gastric mucosa. Stagrration of gastric contents leads to bacterial
action and formation. Fermentation of CHO by lactic acid forming bacteria (Boas Oppler
Baciltus). Lactic acid in gastric juice:

a) bacterial fermentation
b) gastric carcincma

c) ingestion of sour mitk


3. Blood in'gastric contents:
a) may bd due to lesions in the mouth respiralory tract and nasopharynx, Gl tract
b) gastric carcinoma
c) peptic ulcer
4) Detection of Occult Blood: .
Deep blue for 3 secs- indicate 75 % blood . Pale blue for 4- 15 seconds - indicates 1-5% blood
Pale blue lasting for 15-30 seconds- indicates < 1 o/o blood
ACTIVITY NO 25
FECES
FECES
Waste matter discharged from the intestine; excrement

fermentation of

CHO which were


inadequately digested/ absorbed/
unabsorbed fatty acids. Found in highly
acidic feces.

Form and Consistency


Normal: sofl and formed

Abnormal:

- soft and watery (diarrhea)


- exccssively hard stools (constipation)
' - gaseous (fermentive) slools- soft and mushy.
Bubbles of gas may be p,esent which become
more evident after the stools stand in warm
place for 12 hours. Due to excessive CHO
fermentation.

consists of undigested food remnanls, various products of digestion, enormous no. of


bacteria, most if not allare non pathogenic.Patient with diarrhea, has increased food
remnantsdue to its passage through the intestine before drgestion and absorption
are complete. (Digestionimoaired).

- Flattened/ ribbon like- due to obstruction in

Macroscopic Examination
l. Color
Normal: lighVdark browndue to stercobilin
Abnormal:

varices in cirrhosis of liver, carcinoma of Gl tract


and hemonhoids.

Yellow- due to milk diet. corn meal , fats


Green- due to spinach, green leafy vegetables..

lower portion of colon

IV. Blood
- most common caused by ulcers, esophageal
- The higher the origin of biood in Gl tract, the
darker and more thoroughly mixed it will be with
feces.

Bright red- bleeding in the intestinal tracl,

Detection of Occult Blood

undigested beets or tomatoes

- green to blue color (+ blood)

Dark redl chocolate brown- excess coffee,


cocoa, chocolate, black benies etc.

Black- after taking Fe, bismuth suboxide, or


charcoal or due to digested blood.

Normal: foul/ pungent due to indole, skatole and


butyric acid.

Detection of Bilirubin (Gmelin's Test)


Bilirubin- pigment produced from the breakdown
of hemoglobin and secreted into bile.

+ reaction: play of colors: glden on peripher!, '"


toward center blue, violet, red and yellow

. Absence of green excludes the preserrce of bile

Abnormal:

. Extremely foul- indicatcs putrefaction due to


undiocsled nrOtein.
2. Putrid- found in ulceraied and malignant
tumors of the lower bowel and in large
1

u,,v,vvvtvv Hl

hemorrhages.

3. Sour and rancid- indicates gas formation and

prgments.

F. Detection of Urobilin/ Stercobilin (Smith's Test)


- brownish yellow
Answers to Questions:
1. Presence of excessive amounts of bilirubin in feces

indicates the rapid passage of feces in the large


intestine giving no time for bilirubin to be transformed
into stercobilin.
- lncreased bilirubin prevents formation of stercobilin
which makes feces darker in color.
2. Significance of the total absence of bile pigments in
feces:
- obsiruction to the flow of bile/ suppression of bile
pigments from liver.

- Steatorrhea- excessive faV lipid in feces.


3. Stools darken upon exposure due to colorless
stercobilinogen is oxidized to stercobilin.
4. Color is chiefly due to stercobilin. lt depends also on
diet.
5. Odor of stool is due to:

. HzS- from deamination of cysteine


o Indole and skatole- deamination of TRP
. High protein and CHO intake
e Slightly sour odor of infant feces due to
organic acids (lactic, butyric and acetic)
resulling from bactclial fenncrrtation of CHO.
6. Consistency of feces:
Depend on:

. Nature of diet
Example:
Rancid food- very sofl, watery/ ribbon like
High lipid- foamy and mushy
Thoroughly digested- hard and dry
Vegetarian- soft stool
Water intake and i-eabsorotion in colon.