POST LAB EXPT 4

Enzymes

• mostly protein molecules that act as catalysts for biochemical reaction

• very specific; each enzyme catalyzes a certain reaction or type of reaction only

Catalysts

increase the rate of reaction without changing the free energy

Oxidoreductase

catalyzes redox reactions

Transferase

catalyzes the transfer of a specific chemical group from one molecule to another

Ligase

catalyzes the joining of 2 molecules

Hydrolase

catalyzes the breaking of bonds within a molecule by using water

Lyase

catalyzes the breaking of certain chemical bonds in molecules through mechanism other than hydrolysis or oxidation

Isomerase

catalyzes the conversion of a molecule into its isomer (rearrangement)

Active site

an enzyme contains an … that binds the substrate, forming an enzyme-substrate complex

Substrate

reactant; substance upon which the enzyme acts

Products

once the reaction has occurred, the catalyst releases the products

Life would not be possible

without enzymes, … since biochemical reactions would not occur as fast as they would

Enzyme activity

• refers to the measure of the rate at which enzyme converts substrate to products in a biochemical reaction

Factors affecting enzyme activity

• temperature

• pH level

• substrate concentration

• enzyme concentration

• presence of inhibitors

Objectives

• learn the activity of salivary amylase in the digestion of carbohydrates

• determine the effects of enzyme concentration in enzyme activity

• determine the effects of pH in enzyme activity

• determine the effects of inhibitors

Methodology

• collection of saliva sample

• subjecting samples to different conditions

• dropping samples in a spot plate for iodine test

• observation color changes at different time points

Effect of enzyme concentration (experimental set up)

• test tube 1: contains starch only

• test tube 2: contains 3 drops of saliva

• test tube 3: contains 6 drops of saliva

• test tube 4: contains 10 drops of saliva

Effect of temperature and inhibitors (experimental set up)

• test tube 1: ice bath

• test tube 2: 37C

• test tube 3: 100C

• test tube A: +NaCl

• test tube B: +EtOH

• test tube C: +AgNO3s

Iodine test

Iodine reacts with starch to form a starch/iodine complex which gives a characteristic blue color to the reaction mixture

Practical iodine test application

iodine testing to analyze fruit ripening process (plant biology) and identify adequate dietary intake or overload of iodine (nutrition)

Dark blue back

• amount of starch remaining: all

• enzyme activity level: none (0)

Blue

• amount of starch remaining: most

• enzyme activity level: low (1)

Light brown

• amount of starch remaining: some

• enzyme activity level: moderate (2)

Gold

• amount of starch remaining: none

• enzyme activity level: high (3)

Starch

polysaccharide made up of glucose units and consists of 2 main components—amylose and amylopectin

Iodine

• not very soluble in water as itself but forms triiodide (I3-) or pentaiodide (i5-) ions that slip into the helical structure of amylose

  • this causes a visible color change (characteristic blue black) indicative of the presence of starch in the samples

Effect of enzyme concentration

• the rate of an enzyme catalyzed reaction increases with an increase in the concentration of an enzyme

  • more drops of saliva sample

  • more salivary amylase to break down starch

  • high enzyme activity

Longer incubation time

allows for more substrate molecule to be converted into product by the enzyme up to a certain point

Prolonged incubation

may also lead to enzyme denaturation (decreases enzymatic activity) due to extended exposure

pH

2, 4, 7, 10

Temperature

• ~10C (ice bath)

• 37C (body temperature)

• 100C (boiling water bath)

a-amylase

• has an optimum pH of 7 (neutral) due to this being the pH of saliva which acts in the beginning of starch digestion

• has an optimum temperature of 32C-37C because this mimics or is close to the normal human body temperature

• deviations (higher or lower values than optimum) from this pH level or temperature may lead to a decrease in enzyme activity possibly either due to less interactions with the substrate or enzyme denaturation

At temperatures lower than the optimum

there may be decreased catalytic activity due to insufficient energy for collisions

At temperature higher than the optimum

enzyme denaturation may happen thus inactivating the enzyme

Small changes in the optimum pH value

can result in denaturation of enzymes since there can be acidic or basic AA residues in the enzyme

Protonation or deprotonation

may inactivate the enzyme

Inhibitors

• 1% NaCl

• 95% ethanol

• AgNO3

Binding of inhibitors to the enzymes

decrease enzyme activity

1% NaCl

• typically used as a reagent in buffer solutions for amylase assays

• does not inhibit enzyme activity also due to its low concentration

95% ethanol

alcohols especially concentrated ethanol, inhibit a-amylase activity by disrupting the secondary structure of the enzyme—making it difficult for the enzyme to bind to and break down starch

AgNO3

• silver nitrate acts as noncompetitive inhibitor of a-amylase by the interaction of the silver ions to the enzyme, altering its active site and preventing its interaction

• also inhibits a-amylase activity by disrupting the secondary structure of the enzyme—making it difficult for the enzyme to bind and break down starch

No biological activity

decreased enzyme activity or enzyme inactivation

Denaturation

• unraveling of the 3D structure of a macromolecule caused by the breakdown of noncovalent interactions that can result from:

  • heat

  • large changes in pH which alter charges on side chains

  • detergents, such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions

  • urea and guanidine hydrochloride, which disrupt hydrogen bonding

  • B-mercaptoethanol