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 energy released by the reaction products starting substance

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 Substrate Enzyme Active site NORMAL BINDING OF SUBSTRATE Competitive inhibitor Non- competitive inhibitor ENZYME INHIBITION

8 Condensation (aka Dehydration Synthesis) Two molecules combine Water is a byproduct

9 Hydrolysis Type of cleavage reaction (opposite of condensation) Most digestive enzymes catalyze hydrolysis reactions. Addition of H 2 O breaks polymers into smaller subunits (monomers, dimers ect..)

10 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

11 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.

12 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

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 13

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

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 1.Brush-border membrane peptidases 2.Brush-border membrane amino acid transporters 3.Brush-border membrane di- and tripeptides transporters 4.Intracellular peptidases 5.Basolateral-membrane amino acid carriers 6.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 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.

triglyderides 22

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

Lipase pH Test Results 25 HO- +H

26 The End