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 reactiono Enzyme is the most efficient catalyst that can increase the rates of reaction

by 1020

GENERAL STRUCTURAL CLASSES:

1) SIMPLE ENZYME Is an enzyme composed of protein (amino acid chains))2) CONJUGATED ENZYME It has protein and non-protein part

o Apoenzyme – is a protein part of a conjugated enzymeo 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 inproteino The Lipases – effect the breaking of ester linkages in triacyglycerols

Example

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 bondo Hydratase – effects the addition of the component of water to a double

bond 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.

MODELS OF ENZYME ACTION

ENZYME ACTIVE SITEo “Crevicelike” location in the enzyme that participates in the in the

interaction with substrate during the reactiono It is small part of an enzyme’s structure that is actually involved in catalysis.

SUBSTRATE o The reactant in an enzyme catalyzed reaction

ENZYME - SUBSTRATE COMPLEXo Is the intermediate reaction species that is formed when a substrate binds

to the active site of an enzyme.

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.

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 activityo Human body has 37 degree Celsius optimum temperature to carry out

reactiono 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

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.

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.

- 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.

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.

Scan…pix pp202-203B. 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.

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 cause’s enzyme inactivation.

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 23SALIVA 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 mucino Solvent for soluble substanceso Diluents for irritant substances like alkalies ans acidso 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 NO

NAME OF THE TEST/REAGENT ADDED

RESULT OBSERVED CONCLUSION/ PRESENCE OF

1 Molisch test Violet ring formation CHO2 Biuret test Purple color 2 peptide3 Benedict’s test Blue No reducing sugar4 Dil. Acetic acid White ppt mucin5 Dil. HNO3+AgNO3 White ppt Cl-6 Dil. HCL+BaCl2 White ppt SO47 Dil. HNO3+ammonium

molybdateYellow ppt PO4

8 Dil. Acetic acid+ (NH4)2C2)4

White ppt Ca++

9 Dil.H2SO4 + KI+starch Darck blue NO210 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 substanceb. Chemical factor – action of sugars, acids and salts in the taste budsc. Exposure to air- saliva lose CO2 and pH rises rapidlyd. 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 CaC2O4d. 2KI+2NO2 2KNO2+2Ie. 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.

ACTIVITY NO. 23PTYALIN

A. 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

Halogens increases enzymatic activity but not in combination with heavy metals and organic compounds

Chlorides, bromides and nitrates increases the activity.

ANSWERS TO QUESTIONS:

Answers to Questions: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-lodide and nitrates- less effective-Sulfate, Acetates anci Phosphates- least effective

ACTIVITY NO 24GASTRIC ANALYSIS

Importance:1) To gain information regarding the secretory and motor activities of the stomach2) Reveal the presence of substances which are indicative of pathological states3) Differential diagnosis of gastric ulces from duodenal ulcer4) Measure amount of acid produced by patient

GASTRIC CONTENT:Gastric- having to do with or near the stomachFunction 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 HCL1) Boas test- red streaks 2) Gunzberg Test – red spotsC. Test For lactic Acid1) Strauss Test – 2 layers; ether (green); water (yellow)2) Uffelman’s test – yellow brown / light brown colorD. 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 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 protective mucus.

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% bloodPale blue lasting for 15-30 seconds- indicates < 1 o/o blood

ACTIVITY NO 25FECES

FECES Waste matter discharged from the intestine; excrement

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).

Macroscopic Examinationl. ColorNormal: lighVdark browndue to stercobilinAbnormal:Yellow- due to milk diet. corn meal , fatsGreen- due to spinach, green leafy vegetables..

Bright red- bleeding in the intestinal tracl,undigested beets or tomatoes

Dark redl chocolate brown- excess coffee,cocoa, chocolate, black benies etc.

Black- after taking Fe, bismuth suboxide, orcharcoal or due to digested blood.

Normal: foul/ pungent due to indole, skatole andbutyric acid.Abnormal:

1 . Extremely foul- indicatcs putrefaction due to

undiocsled nrOtein. u,,v,vvvtvv Hl 2. Putrid- found in ulceraied and malignant

tumors of the lower bowel and in largehemorrhages.

3. Sour and rancid- indicates gas formation and

fermentation of CHO which were

inadequately digested/ absorbed/

unabsorbed fatty acids. Found in highlyacidic feces.Form and ConsistencyNormal: sofl and formedAbnormal:

- soft and watery (diarrhea)

- exccssively hard stools (constipation)

' - gaseous (fermentive) slools- soft and mushy.Bubbles of gas may be p,esent which becomemore evident after the stools stand in warm

place for 12 hours. Due to excessive CHOfermentation.

- Flattened/ ribbon like- due to obstruction inlower portion of colon

IV. Blood - most common caused by ulcers, esophagealvarices in cirrhosis of liver, carcinoma of Gl tractand hemonhoids.

- The higher the origin of biood in Gl tract, thedarker and more thoroughly mixed it will be withfeces.

Detection of Occult Blood

- green to blue color (+ blood)Detection of Bilirubin (Gmelin's Test)Bilirubin- pigment produced from the breakdownof 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

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 largeintestine giving no time for bilirubin to be transformedinto stercobilin.- lncreased bilirubin prevents formation of stercobilinwhich makes feces darker in color.2. Significance of the total absence of bile pigments infeces:

- obsiruction to the flow of bile/ suppression of bilepigments from liver.

- Steatorrhea- excessive faV lipid in feces.

3. Stools darken upon exposure due to colorlessstercobilinogen is oxidized to stercobilin.4. Color is chiefly due to stercobilin. lt depends also ondiet.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 toorganic acids (lactic, butyric and acetic)resulling from bactclial fenncrrtation of CHO.6. Consistency of feces:Depend on:

. Nature of dietExample:Rancid food- very sofl, watery/ ribbon likeHigh lipid- foamy and mushyThoroughly digested- hard and dryVegetarian- soft stoolWater intake and i-eabsorotion in colon.