Lab Activity 30 Digestive Enzymes Portland Community College

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. 2

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 3

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… 4

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

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 6

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 alters an enzyme’s function by changing its shape ENZYME INHIBITION 7

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

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. . ) 1 1 2 2 3 4 3 9

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 10

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. 11

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 12

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 p. H change 13

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

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! 15

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

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) 17

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 18

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 19

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!!!) 20

Fat Digestion • Goal #1: Emulsify big fat globules O O O into tiny fat droplet spheres ooooooo • 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. 21

triglyderides 22

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) 23

Litmus Cream • Litmus is a p. H 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 p. H, and the litmus solution turns light PINK 24

Lipase p. H Test Results HO+H 25

The End 26