Download Ativity 30

Lab Activity 30
Digestive Enzymes
Portland Community College
BI 233
Cellular Reactions
• All molecular bonds have energy barriers that
prevent spontaneous breakdown
• Enzymes lowering these “activation energy”
barriers; the enzyme reduces the energy that must
be absorbed by the reactants
• This allows the reaction to progress (to
equilibrium) rapidly even at a the relatively low
temperature of your body.
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Energy of Activation (EA)
• For a reaction to occur, an
energy barrier must be
overcome.
• Enzymes make the
energy barrier smaller
• (Imagine a train passing
through a tunnel .)
• Enzymes do not make a
non-spontaneous
reaction spontaneous.
EA
without
enzyme
starting
substance
EA with
enzyme
energy
released
by the
reaction
products
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Enzymes
• …are proteins – biological catalysts that lower the
activation energy of a reaction.
• …are highly specific; they only act only on a small
number of substrates (often just one.)
• …increase the rate of a chemical reaction.
• …are re-used; they are not consumed in the reaction.
E + S ES complex  E + Product(s)
*If there is no working enzyme, the reaction may still
occur very slowly, eventually…
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Enzymes
• Environmental conditions affect enzymes:
• Temperature
• pH
• Salt concentration
• When you “denature” an enzyme, you change its
shape
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Enzyme Helpers
• Some enzymes require non-protein cofactors
• Some are inorganic metal ions of zinc, iron,
and other trace elements
• Some are organic molecules called
coenzymes
• Includes vitamins or altered vitamin
components
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Enzyme Inhibitors
• Inhibitors block
enzyme action
NORMAL BINDING OF SUBSTRATE
Substrate
Active
site
• A competitive
inhibitor takes
Enzyme
the place of a
substrate in the
Nonactive site
Competitive
competitive
• A noncompetitive inhibitor
inhibitor
inhibitor alters an
enzyme’s function
by changing its shape
ENZYME
INHIBITION
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Condensation
(aka Dehydration Synthesis)
• Two molecules combine
• Water is a byproduct
1
2
1
3
2
3
4
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Hydrolysis
• Type of cleavage reaction (opposite of
condensation)
• Most digestive
enzymes catalyze
hydrolysis reactions.
• Addition of H2O
breaks polymers into
smaller subunits
(monomers, dimers
ect..)
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1
2
2
3
4
3
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Four types of Macromolecules
Class
Monomer(s) Polymer(s)
Carbohydrates
monosaccharides
polysaccharides
Proteins
amino acids
polypeptides
Lipids
fatty acids
and glycerol
triglycerides,
phospholipids,
steroids*
polynucleotides
Nucleic acids
nucleotides
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Carbohydrate Digestion
•
Goal #1: Break complex carbs (starch)
down to oligosaccharides, trisaccharides,
disaccharides
1. Salivary Amylase: (minor): breaks complex
carbs (starch, glycogen) to oligosaccharides,
trisaccharides, and disaccharides. Inactivated by
gastric acid.
2. Pancreatic amylase: (major)
3. Amylase is also in breast milk.
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Carbohydrate Digestion
• Goal #2: further breakdown into
monosaccharides
• Use brush border enzymes on microvilli of
small intestine
1. Lactase: breaks lactose into glucose + galactose
2. Maltase: breaks maltose into 2 glucoses, (also works
on oligosaccharides)
3. Sucrase: breaks sucrose into glucose + fructose
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Introduction to
Four Diagnostic Tests
• Lugol’s IKI test
• Color change indicates presence of starch
• Benedict’s Solution test
• Color change (with heat) indicates presence of
glucose or maltose
• BAPNA test
• Color change indicates enzyme activity
• Litmus Cream (or Litmus Paper) test
• Color change indicates pH change
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Lugol’s IKI
• IKI: potassium iodide
• Turns black in the presence of starch
IKI alone
Positive result
(yes, starch!)
Negative result
(no starch)
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Benedict’s Solution
• Benedict's solution allows us to detect
glucose (Glc) and maltose (Glc-Glc)
• It is a blue solution that will turn red-orange
(yield brick red solid precipitate) when heated
in the presence of glucose or maltose.
• Note that sucrose (Glc-Frc) will not trigger a
color change!
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Benedict’s
Solution
Before heating
(All start blue.)
After heating
(“Orange is
positive.”)
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Protein Digestion
• Goal #1: Break peptide bonds of proteins to
yield smaller polypeptides
• HCL in stomach first denatures the proteins to
enhance chemical digestion by exposing
peptide bonds.
• Enzymes break peptide bonds between amino
acids of proteins to make smaller polypeptides
• In stomach: pepsin (from pepsinogen from the
stomach’s chief cells)
• In intestine: pancreatic enzymes (trypsin, elastase,
chymotrypsin & carboxypeptidase)
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Protein Digestion
• Goal #2: break small polypeptides into
single amino acids.
• Enzymes:
• On brush border: peptidases
• Inside cytoplasm of intestinal cells: several
dipeptidases, tripeptidase break absorbed
dipeptides and tripeptides into amino acids
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Protein
Digestion
1. Brush-border membrane
peptidases
2. Brush-border membrane
amino acid transporters
3. Brush-border membrane diand tripeptides transporters
4. Intracellular peptidases
5. Basolateral-membrane amino
acid carriers
6. Basolateral membrane di- and
tripeptides carriers
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Protein Digestion
• BAPNA is a color-changing dye that is
attached to an amino acid via a peptide
bond.
• Review: peptide bonds link amino acids in the
proteins (polypeptides) that you eat.
• When BAPNA’s peptide bond is broken (using
an enzyme such as trypsin,) the dye is released
and it turns from clear to yellow. (Don’t drink
the BAPNA!!!)
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Fat Digestion
• Goal #1: Emulsify big fat globules O O O
into tiny fat droplet spheres
oooooooooooooo
• Bile salts emulsify
• Lipase (a water soluble enzyme that can’t
penetrate fat droplet) will efficiently react with
surface fat
• Smaller spheres of fat have higher surface/volume
ratio, so the lapse can work faster on many small
droplets than on one large globule.
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triglyderides
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Fat Digestion
• Goal #2: Break triglycerides into monoglycerides
and fatty acids
• yields monoglycerides and fatty acids
• chief cells in fundus : gastric lipase
• about 20% of fat digestion
• intestines: pancreatic lipase
• about 80% of fat digestion
• breast milk: milk-derived lipase
• yields fatty acids and glycerol
(not fatty acids and monoglycerides)
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Litmus Cream
• Litmus is a pH indicator
• purple in storage bottle, it may turn to dark
lavender or light pink
• It comes mixed with cream (a source of
triglycerides!)
• Triglyceride digestion by lipase releases
fatty acids.
• These fatty acids drop the pH, and the litmus
solution turns light PINK
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Lipase pH Test Results
HO+H
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The End
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