Salve Marie Fernandez BS Biology III Exercise 6

Enzyme
They are biological catalysts that increase the rate of reactions and are highly specific. Functionally they regulate metabolic processes in the cell without itself being affected. A. Hydrolases
a. Amylase Iodine test of 0.1% starch solution After Incubation with corn Light blue color Colorless/light blue Benedicts solution with 1% Without corn sucrose b. Sucrase with corn Light color (no ppt) Bricked red ppt Amylase enzyme breaks down starches into simpler forms which is soluble in

water. Amylose present in starch is responsible for the formation of a deep blue

color in the presence of iodine. The iodine molecule slips inside of the amylose coil. The absence of dark blue color suggests that starch had been broken down due to the presence of amylase enzyme. Under optimum temp, room temperature, the enzyme works best. Benedict's reagent is used as a test for the presence of reducing sugars. If reducing sugar is present the color of the solution will produce a brick red precipitate. Sucrase is the name given to an enzyme that catalyze the hydrolysis of sucrose to fructose and glucose. Starch had been broken down due to the presence of sucrose enzyme that resulted to a brick red ppt indicating the presence of reducing sugars. B. Oxidoreductases
With .001% methylene blue a. Dehydrogenases w/ germinated munbean : colorless w/o germinated mungbean: blue After aeration Theoretica Result l result obtained blue colorless blue blue

Methylene blue is blue when oxidized, but turns colorless when reduced. Methylene blue can, therefore, be used to show the presence of active dehydrogenase enzymes by a color change. Dehydrogenase enzymes remove hydrogen from their substrate. As a result oxygen is liberated and is free for take up of the seedling. Methylene blue isreduced and seed gets its needed oxygen. Presence of dehydrogenase in germinating munbean seed reduced the methylene

blue solution in a closed set up. When aerated methylene blue solution turns to blue again.

b.

Catalase

Unboiled preparation Bubble formation/gas evolution

boiled preparation No bubble formation/ gas evolution

Catalase enzyme present in potatoes catalyzes the decomposition of hydrogen peroxide to water and oxygen. Boiling causes the denaturation of the enzyme that inactivates the enzyme activity thus; it can no longer catalyze the breaking down of H2O2 to water and oxygen. That would produce no evolution of gas/bubbles.

2H2O2 2 H2O +O2

C. Factors affecting enzyme activity

Test tube #1 (4ml amylase) colorless Test tube # 2 (2ml amylase:1ml H2O) blue solution Test tube #3 (1ml amylase:3ml H2O) Relatively darker blue

a. Enzyme concentration

An increase in enzyme concentration will increase the enzyme reaction rate until the substrate becomes limiting. Results show that test tube 1 having the most concentrated amount of amylase had reacted to the starch solution significantly that resulted to a colorless solution. Test tube 3 having the most diluted enzyme concentration had lesser enzymatic reaction with the starch solution producing a dark blue color. Starch solution with 5 ml buffer solutions and 2ml amylase b. Hydrogen ion enzyme concentration pH 4 pH 7 pH 10 dark colorless dark An enzyme has its optimal pH that helps maintain its three dimensional shape. Changes in pH maydenature enzymes by altering the enzymatic charge. This alters the ionic bonds of the enzyme that contributes to its functional shape. Amylase enzyme works best at pH 7. 5-10 C 28-30 C 98- 100 c. Temperature dark Colorless Dark (+)for starch (-) for starch (+) starch Enzyme has also its optimum temperature where it works best. As temperature increases, molecular motion increases resulting in more molecular collisions. If temperature rises above optimum point, heat will denature enzyme that would disrupt its natural state by denaturing hydrogen bonds. Low temperature also slows down enzyme activity by decreasing molecular motion. Enzyme works best at 28 - 30 C. The farther the temperature from its optimum point, the lesser the enzymatic activity. Guide questions: 1. a. Pyruvate + NAD + + COA Acetyl CoA + NADH + H+ + CO2 (i) Pyruvate dehydrogenase (ii) MItochondria

(iii) Krebs cycle/ Citric Acid cycle - Respiration b. Ribulose 1, 5 biphosphate + CO2 2(3-phosphoglyceri acid) (i) Ribulose bisphosphate carboxylase oxygenase (RuBisCO) (ii)Stroma of Chloroplasts (iii) Carbon fixnthesisation of Calvin cycle - Photosy c. Fructose -6- phosphate + ATP Fructose -1, 6 biphosphate (i) Phosphofructokinase (ii) cytsol (iii)Glycolysis

2. At a low substrate concentration and constant enzyme concentration there are many active sites that are not occupied thus reaction rate is low and the substrate becomes the limiting factor. When more substrate molecules are added, more enzyme substrate complexes can be formed; rate of reaction increases. Increasing the substrate concentration further will have no effect, the active sites will be saturated and no more enzyme substrate complexes can be formed. 3. At enzymes optimum pH, the shape of the enzyme is such that the active site can fit perfectly with the substrate. As pH decreases from, or increases from the optimum, the acid or base conditions begin to disrupt some of the hydrogen bonds between loops of the protein chains. The active site will be disrupted and the enzyme denatured.