Lab 5: Enzyme Function

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Lab 5 Enzyme Function
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Introduction

• ENZYME an organic catalyst, which speeds up the
  rates of chemical reactions in living systems.
• CATALYST any substance which affects the rate
  of a chemical reaction without itself being used
  up.

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Introduction (cont.)

• All but 2 known enzymes are proteins.
• To understand how enzymes function, you must
  understand the basic structure of proteins.

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I. Proteins

• Proteins are composed of amino acids (AA).
• In adult humans, there are eight naturally
  occurring AAs that cannot be synthesized in the
  body. These essential amino acids must be
  obtained from the food of the organism.

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I. Proteins

• Amino acids contain a central Carbon atom which
  is bonded to
• a Hydrogen atom
• -H
• amino functional group
• -NH2
• carboxyl functional group
• -COOH
• R group ( rest of molecule), which determines
  the different biological properties of individual
  amino acids.

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I. Proteins (cont.)

• Three different R-groups makes 3 different amino
  acids.

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I. Proteins (cont.)

• Amino acids, which make up proteins, are linked
  together by peptide bonds.
• The process involves the union of an amino group
  and a carboxyl group, resulting in the loss of a
  water molecule ( Dehydration Synthesis Reaction)

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I. Proteins (cont.)

• Dipeptide 2 AA linked together
• Tripeptide 3 AA linked together
• Polypeptide gt 3 AA linked together
• Proteins may range in length from a few hundred
  to more than a thousand amino acids.

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I. Proteins (cont.) Levels of Protein structure

• Primary (1) structure a sequence of amino
  acids in polypeptide chain.
• The sequence of the AA determines overall 3-D
  structure function of proteins.

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I. Proteins (cont.) Levels of Protein structure

• Two common secondary (2) structures
• helix - stretchable coils
• pleated sheet - non-elastic sheets

H bonds
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I. Proteins (cont.) Levels of Protein structure

• Tertiary (3) Structures
• Results from folding of 2 structure
• due to bonding between R groups of amino acids

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I. Proteins (cont.) Levels of Protein structure
Quaternary (4) Structures gt2 peptide chain
subunits Example Hemoglobin is composed of 4
polypeptide chains, each with its own 1, 2, and
3 structures.
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Video Animation Levels of Protein Organization
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I. Proteins (cont.) Protein Denaturation

• Protein denaturation a protein unravels and
  looses its 2 or 3 structure.
• results in loss of protein function.
• denaturation can be reversible.

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I. Proteins (cont.) Protein Denaturation

• Unfolding of polypeptide chains loss
  of biological activity (either permanently or
  temporarily).

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I. Proteins (cont.) Protein Denaturation

• Causes of protein denaturation include 1.
  Excessive heat few proteins can tolerate
  temperatures above 60 celsius. The 3 structure
  is distorted. example the white of an egg
  becomes opaque when cooked, because the proteins
  are denatured by the heat.
• 2. extreme (high or low) pH
• 3. high salt concentration

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Video Animation How Enzymes Function
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II. Enzymes

• Enzymes speed up biochemical reactions by
  lowering the activation energy ( amount of
  energy reactants must absorb to start the
  reaction).

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II. Enzymes (cont.) Factors affecting rate of
enzyme activity

• A. Temperature

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II. Enzymes (cont.) Factors affecting rate of
enzyme activity
Increase temperature increase rate
enzyme activity. The reaction is faster because
rate at which enzymes substrate collide is
increased.
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Enzymes have optimum temp. for catalyzing
reactions
a. The optimal temperature for activity of most
human enzymes 35-45 oC b. The optimal
temperature for enzymes of hot springs bacteria
(Archeae) 70 oC
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II. Enzymes (cont.) Factors affecting rate of
enzyme activity

• Above the optimal temperature range, secondary
  and tertiary protein structures are altered, the
  active sites destroyed, and the function of the
  enzyme is lost.
• Below the optimal temperature range, the enzyme
  reaction rate is decreased, because the enzyme is
  coming into contact with the substrate at a
  slower rate.

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B. pH
1. pH a measure of acidity or alakalinity of a
solution
2. Acids Bases
a. Acids substances which cause increase in
H in solution
b. Bases substances which cause decrease H
in soln. increase OH- in solution
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3. pH Scale measure of relative H OH-
values of 0 - 14
Acid
a. pH below 7 - H greater than OH- _____
b. pH 7 - H equals OH- Neutral
Base
c. pH above 7 - H less than OH- ____
d . pH scale is logarithmic, therefore, 1 pH
unit 10 fold change in H.
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4. Too high or low H distorts weak chemical
bonds responsible for 2o 3o structure of
enzymes altered active site
loss enzyme activity
5. Each enzymes has its own pH optimum.
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pH 6 - 8
a. enzymes in most human cells
b. pepsin (stomach dig. enz)
pH 2
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Experiment One The effect of Temperature pH on
Bromelain Activity
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I. Biology of Pineapple
The cluster of flowers resembles a pine cone,
hence the name.
Leaves
The pineapple is considered a multiple
fruit, Why?
Each "section" develops fromthe ovary of 1 flower
Anansus comosus
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Cells in fruit, leaves, stem contain the enzyme
bromelain
Bromelain is a protease
Protease
Suffix meaning enzyme (which acts on substrate)
The substrate acted upon (protein)
An Enzyme that catalyzes the digestion
(hydrolysis) of proteins into their amino acid
building blocks.
bromelain
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To determine the rate at which bromelain
catalyzes the digestion of gelatin we
have two options.
1. Measure the rate at which the products (amino
acids) are produced as a result of the digestion
reaction.
2. Measure the rate at which the substrate
(gelatin) is digested.
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Assay for bromelain activity will measure the
rate at which the substrate (gelatin) is
digested. When the enzyme is working, the
gelatin should be disgested, and the black film
strip should clear.
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Write down your hypothesis as to the effect of
temperature pH on the rate of bromelain
activity. Think Will the rate of the
bromelain catalyzed reaction increase, decrease,
or remain the same as the temperature
increases? AND Will there be a lower and upper
temperature limit to this range?
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Effect of Temperature on Enzyme Activity

• Person 1 - 20C and 70Celsius
• Person 2 - 50C
• Person 3 - 40C
• Person 4 - 30C

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Effect of Bromelain Enzyme on Gelation

• As a Table of Four

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Effect of pH on Enzyme Activity

• Pair 1 Test tubes 1-6
• Pair 2 Test tubes 7-10

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Part II Effect of Bromelain on Gelation
Canned
Hot (Fresh)
Fresh



Gelation () No Gelation (-)?
Gelation () No Gelation (-)?
Gelation () No Gelation (-)?
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Bromelain in the Market Place!
Does it Work?
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The Debate after concluding your experiments,
which side do you believe?
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