Lab Activity 30

1. Lab Activity 30 Digestive Enzymes Portland Community College BI 233

2. 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.

3. 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 EA with enzyme starting substance energy released by the reaction products

4. 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…

5. Enzymes • Environmental conditions affect enzymes: • Temperature • pH • Salt concentration • When you “denature” an enzyme, you change its shape

6. 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

7. 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 NORMAL BINDING OF SUBSTRATE Substrate Active site Enzyme Non-competitiveinhibitor Competitiveinhibitor ENZYME INHIBITION

8. Condensation (aka Dehydration Synthesis) • Two molecules combine • Water is a byproduct 1 2 3 1 2 3 4

9. 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..) 1 2 3 4 2 1 3

10. Four types of Macromolecules Monomer(s) Polymer(s) Class Carbohydrates monosaccharides polysaccharides Proteins amino acids polypeptides triglycerides, phospholipids,steroids* fatty acids and glycerol Lipids Nucleic acids nucleotides polynucleotides

11. Carbohydrate Digestion • Goal #1: Break complex carbs (starch) down to oligosaccharides, trisaccharides, disaccharides • Salivary Amylase: (minor): breaks complex carbs (starch, glycogen) to oligosaccharides, trisaccharides, and disaccharides. Inactivated by gastric acid. • Pancreatic amylase: (major) • Amylase is also in breast milk.

12. Carbohydrate Digestion • Goal #2: further breakdown into monosaccharides • Use brush border enzymes on microvilli of small intestine • Lactase: breaks lactose into glucose + galactose • Maltase: breaks maltose into 2 glucoses, (also works on oligosaccharides) • Sucrase: breaks sucrose into glucose + fructose

13. 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

14. Lugol’s IKI • IKI: potassium iodide • Turns black in the presence of starch Positive result (yes, starch!) Negative result (no starch) IKI alone

15. 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!

16. Benedict’s Solution Before heating (All start blue.) After heating (“Orange is positive.”)

17. 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)

18. 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

19. Protein Digestion • Brush-border membrane peptidases • Brush-border membrane amino acid transporters • Brush-border membrane di- and tripeptides transporters • Intracellular peptidases • Basolateral-membrane amino acid carriers • Basolateral membrane di- and tripeptides carriers

20. 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!!!)

21. 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.

22. triglyderides

23. 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)

24. 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

25. Lipase pH Test Results +H HO-

26. The End The End