MODULE 5: ENZYMOLOGY

Module Coordinator: Floro Madarcos, MD

Laboratory Coordinator: Karen M. Faustino, MD

Module Overview:
The introduction to water, buffers and basic acid base balance is a very important concept that governs the
normal function and processes of the body. We will utilize the following teaching learning activities: a case with guide
question, a recorded/ annotated lecture on the concepts and its application, worksheets and a laboratory activity. One
of the activities is a preparation of individual modified Pechakucha 10/10 which will contain 10 slides at 10 seconds per
slide as a summary of the important concepts on water. It will be a graded activity. The synchronous activity will be
the random selection of Pechakucha. It will serve as discussion points during the activity.

Outcomes Content Teaching Learning

activities

At the end of the session, 1. Enzymes, apoenzyme, holoenzyme, Asynchronous activity

the students will be able to: metalloenzyme, regulatory enzyme, active and 1. Power points
allosteric site of an enzyme, isoenzyme,
1. Characterize the six 2. Cases on the
substrate.
major classes of enzymes. application of water and
2. Coenzymes (Vit. B-derived) and inorganic computations
2. Discuss the ions
characteristics of enzymes. 3. Work sheet on
3. Oxidoreductases, Transferases, Enzymes
3. Discuss the enzyme co- Hydrolases, Lyases, Isomerases and Ligases
factors. 4. Formative Quiz
4.Basic enzyme characteristics
4. Describe the models of Synchronous:
enzyme- substrate 5. Lock and Key & Induced Fit Models
Discussion of the
complex.
6. Enzyme kinetics – substrate binding, worksheet and quiz thru
5. Elucidate the kinetics of catalytic step, Michaelis-Menten Equation, zoom
enzyme- catalyzed Michaelis-Menten Curve, Lineweaver Burke ,
reactions. Double Reciprocal Plot . Michaelis constant
(Km) and its significance
6. Describe the operation Laboratory:
and plots used to illustrate 7.Kinetic order of reactions
enzyme kinetics. 8. Reversible and irreversible inhibition • Salivary
9. Feedback inhibition, allosteric and covalent Amylase:
7. Classify enzyme modification, zymogen activation, induction video link to be
inhibition and its effect on and repression of synthesis of key enzymes given
reaction velocity.
10. Temperature, pH, substrate concentration,
8. Discuss the different Cofactors • Interpretation
ways of regulating enzyme 11. Representative drugs that inhibit enzymes of results
activity. Cardiac enzymes as markers for myocardial
infarction
 9. Explain the factors • Guide
affecting enzyme activity. Questions to
be answered
10. Outline the role of
some enzymes used in
• Plenary
clinical diagnosis.
session thru
Google Meet/
zoom 1 hours

WORKSHEET ON ENZYMOLOGY AND PHARMACOTHERAPY

Learning Objectives:
1. Relate enzyme inhibition by certain drugs in the treatment of some clinical
disorders.
2. Classify the enzymes.
3. Identify the chemical groups transferred by coenzymes.
4. Describe the general mechanism of action of enzymes.
5. Differentiate between competitive and noncompetitive inhibition.
6. Elucidate on the factors affecting enzyme activity.
7. State the significance of Km.
8. Describe the 2 plots used in saturation kinetics.

Task I. By means of words and arrows, illustrate and explain the reaction
pathway by which drugs inhibit enzymes to treat diseases:
a. ACE inhibitors for hypertension
b. Statins for hypercholesterolemia
c. Sildenafil for erectile dysfunction
d. Allopurinol for gouty arthritis
e. Methotrexate for uterine cancer
f. Antabuse for chronic alcoholism

Task II: Complete the following table on the enzymes:

Enzyme Classification Coenzyme (if any) Chemical group
transferred by the
coenzyme
Angiotensin converting
enzyme (ACE)
HMG CoA reductase
cGMP
phosphodiesterase
Xanthine oxidase
Dihydrofolate reductase
Aldehyde
dehydrogenase
Task III. Draw and describe the reaction coordinate diagram to explain the
mechanism of action of an enzyme.

Task IV. Complete the following table on the common types of enzyme
inhibition:
Criteria Competitive Noncompetitive
Acting on
Structure of inhibitor
Effect of excess substrate
Effect on Km
Effect on Vmax
Significance/clinical use

Task V. A bacterial enzyme has the following temperature activity profile:

Explain briefly the observed activity or velocity at points:

A.
B.
C.

Task VI. Given a saturation curve, with a plot of reaction velocity (VO) with
increasing substrate concentration [S] for a given quantity of a simple,
nonallosteric enzyme:
 1. Differentiate between an allosteric and a nonallosteric enzyme.
2. Approximate the percentage amount of enzyme-substrate complexes at
points:
A.
B.
C.
3. Explain the following:
a. First order kinetic reaction at Points A & B
b. Zero order kinetic reaction at Point C
4. What is the significance/function of Km of enzymes in general?
5. What is the advantage of the Lineweaver Burke Plot over the Michaelis-
Menten saturation curve in terms of Km and Vmax?

Laboratory
Effect of pH, Temperature, Ions and Substrate Concentration to Salivary Amylase activity

Introduction

The alpha amylases of human saliva, plant tissues and animal pancreatic juices catalyze the rapid, random hydrolysis
or specific internal glycosidic linkages in certain polysaccharides. Salivary amylase progressively hydrolyzes some
polysaccharide into smaller polymerase oligosaccharides, then finally into monosaccharides and disaccharides.

In a glycosidic bond, the aldehyde group of one monosaccharide is linked to a hydroxyl group of the adjacent
monosaccharide. Thus, for every glycosidic bond hydrolyzed by amylase, one aldehyde group is liberated. Since
aldehyde groups are good reducing agents, the progress of hydrolysis can be observed by measuring the increase in
reducing groups as assayed by 3, 5-dinitrosalicylate (DNS) reduction.

In this experiment, we will analyze the substrate specificity and activity of Alpha amylase. We will also investigate how
the enzyme’s activity is affected by pH, temperature, substrate concentration and presence of specific ions. In addition,
the condition required for catalysis by the enzyme will be compared to the requirements in catalysis by HCl.
Reagents and Materials
Saliva Deionized water
1 big beaker Distilled Water
2 small beakers Cold distilled water
1 10ml pipette 0.05M Phosphate Buffer Solutions pH 6.2
2 big test tubes 0.05M Phosphate Buffer Solutions pH 6.7
21 small test tubes 0.05M Phosphate Buffer Solutions pH 7.2
Aluminum foil 0.05M Phosphate Buffer Solutions pH 7.7
 pH paper 0.05M Phosphate Buffer Solutions pH 8.2
DNS reagent glucose standard curve
1% starch solution
2% starch solution

Glucose Standard Curve

0.8
0.7
0.6
absorbance

0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5
mg% glucose

Protocol
1. Preparation of Alpha Amylase solution.
a. Collect 2 ml of saliva in a small beaker. Keep the specimen in an ice batch. This will be the stock
enzyme for all experiments. Note: α-amylase concentration varies among samples of saliva
depending upon the physiological state and genetic constitution of the donor.
b. Prepare a stock saliva solution by adding 0.25 ml of saliva to 19.75 ml of cold distilled H 2O. Keep
diluted saliva in an ice (Be sure to have assay tubes and water bath ready before diluting the saliva).
b1. Boil 1 0ml aliquot of the stock saliva solution over vigorously boiling water for 10
minutes. Cover the test tube with aluminum foil while boiling. Allow to cool and keep in an ice
bath. This will be the boiled saliva.
b2. The remaining 10 ml from the stock saliva solution will be the diluted saliva.
c. Keep the properly labeled 10 ml boiled saliva and 10ml diluted saliva in an ice bath.
 PREPARATION OF ALPHA
AMYLASE SOLUTION
Collect 2ml of saliva – keep in an ice bath

0.25ml saliva aliquot + 19.75ml cold distilled water

(total: 20ml)

10ml aliquot 10ml aliquot

cover with aluminum foil keep in an ice bath
boil over boiling water x 10mins (diluted saliva)
cool then keep in an ice bath
(boiled saliva)

2. Determination of the influence of Temperature to Alpha Amylase Activity (Be sure you have your saliva
solution described in step 1 before starting this segment)

a. Correlate the absorbance with glucose curve above to determine Glucose Concentration.
b. Compute for the enzyme activity (EA=Glucose conc / 20mins). Then plot Temperature vs Enzyme
Activity

3. Determination of the influence of pH to Alpha-Amylase Activity (Be sure you have your saliva solution
described in step 1 before starting this segment)
 a. Correlate the absorbance with glucose curve to determine Glucose Concentration.
b. Compute for the enzyme activity (EA=Glucose conc / 20mins). Then plot pH vs Enzyme Activity

4. Determination of influence of Chloride Ions in Alpha Amylase Activity

a. Compare the two setups. Correlate the absorbance with glucose curve above to determine Glucose
Concentration.
b. Compute for the enzyme activity (EA=Glucose conc / 20mins).. Then compare the enzyme activity
between those 2 tubes (+Cl vs –Cl)

5. Determination of Influence of Substrate Concentration on Alpha Amylase Activity

 a. Correlate the absorbance with glucose curve to determine Glucose Concentration.
b. Compute for the enzyme activity. Then, plot Substrate Concentration vs. Enzyme Activity using
Michaelis Menten plot.
c. Get the reciprocal of the substrate concentration and the reciprocal of the enzyme activity. Then
illustrate the Lineweaver Burke plot.

Name __________________________________ Sec_____ Grp____

Date___________________________
Results

1. Effect of Temperature:
a. Draw a graph representing the effect of temperature on enzyme activity. Indicate the optimum temp
Temperature Absorbance mg% glucose Enzyme Activity
0
40
60
 b. Interpret the graph.

2. Effect of pH
a. Draw a graph representing the effect of pH on Enzyme activity. Indicate the optimum pH.
pH Absorbance mg% glucose Enzyme Activity
6.2
6.7
7.2
7.7
8.2

b. Interpret the graph

 3. Effect of Chloride Ions
a. Compare the amounts of reducing substances produced in the presence and absence of Chloride
ions. Explain the result.
Chloride Absorbance mg% glucose Enzyme activity
Absence
Presence

4. Influence of Substrate Concentration

a. Calculate the amount or reducing substance with the use of the glucose standard curve. The
“preformed” reducing power of the soluble starch is obtained from the glucose equivalent
calculated from tube 7. This is the baseline reducing power for tube 6. The “preformed” reducing
power of the starch present in tubes 1 to 5 can be estimated by ratio and proportion. Subtract the
“preformed” reducing equivalents (mg glucose) from the corresponding values of each tube to
obtain the true glucose equivalent released by the enzyme.
TT [substrate] abs [glucose] Preformed Amount of true reducing Velocity
mg/dl mg/dl glucose glucose equivalent mg/min
mg/dl mg/dl
1 2 A a
2 4 B b
3 5 C c
4 6 D d
5 8 E e
6 10 F f

7 x none

Preformed glucose = by ratio and proportion using [S] TT6/ [G] TT7
Ex: for TT1
10/x= [S]/a 10/x=2/a a= ?
for TT2
10/x= [S]/b 10/x=4/b b= ?

True glucose = [G] – preformed glucose

Ex: for TT1 A-a

Velocity = true glucose/25mins

b. Plot the velocity of the reaction (Amount of reducing substance (mg) formed per minute) vs. the
substrate concentration.

Test tube [substrate] Velocity

(mg/dl) (mg/min)
1 2
2 4
3 5
4 6
5 8
6 10

c. Estimate the value of the Michaelis-Menten constant (Km) from this plot.

d. Calculate the value of Km after plotting the data according to the Lineweaver burke method. Draw
the graph at the back.
Study Guide Questions

1. For the effect of temperature, relate the shape of the curve to the effect of temperature on reaction rates in
general and on enzyme structure.

2. For the effect of pH, determine the optimum pH for salivary amylase activity. Relate the shape of the curve
to the effect of pH on reaction rates in general and on enzyme structure.

3. For the effect of Chloride ion, interpret your data in terms of the possible interaction of Chloride with the
enzyme.

4. For the effect substrate concentration, which is the more accurate method for estimating Km? Why? State
the significance of the Km value obtained.