Lab Activity 30

Digestive Enzymes

Portland Community CollegeBI 233

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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.

EAwithout enzyme

EA with enzyme

energyreleasedby the

reaction

products

starting substance

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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 • A competitive

inhibitor takes the place of a substrate in the active site

• A noncompetitive inhibitor alters an enzyme’s function by changing its shape

Substrate

Enzyme

Active site

NORMAL BINDING OF SUBSTRATE

Competitiveinhibitor

Non-competitiveinhibitor

ENZYME INHIBITION

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Condensation (aka Dehydration Synthesis)

• Two molecules combine• Water is a byproduct

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43

3

2

2

1

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Hydrolysis

• Type of cleavage reaction (opposite of condensation)

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1

2

2 3

3

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• Most digestive enzymes catalyze hydrolysis reactions.

• Addition of H2O breaks polymers into smaller subunits (monomers, dimers ect..)

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Four types of Macromolecules

Carbohydrates

Proteins

Nucleic acids

monosaccharides

amino acids

fatty acids and glycerol

nucleotides

polysaccharides

Monomer(s)

polypeptides

Lipids

Polymer(s)Class

triglycerides, phospholipids,

steroids*polynucleotides

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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 intestine1. Lactase: breaks lactose into glucose + galactose2. Maltase: breaks maltose into 2 glucoses, (also works

on oligosaccharides)3. Sucrase: breaks sucrose into glucose + fructose

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 change13

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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 di-and tripeptides transporters

4. Intracellular peptidases5. Basolateral-membrane amino

acid carriers6. 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 Ointo 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.

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

Lipase pH Test Results

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HO-+H

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The End