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Exercise 6 ENZYMES Results and Discussion Name: Ivy Ann C. Carba Section: HJK Group name: Hale’s Prodigal Daughters Date Performed: Dec. 4-5, 2009 Date Submitted: Dec. 22, 2009 A. Hydrolyses a. Amylase Iodine test of 0.1% starch solution with corn seedling root after incubation → Emergence of a dark purple color. Iodine test of 0.1% starch solution after incubation period (control) → Emergence of a dark purple color. b. Sucrase Benedict’s test of 2% sucrose solution with fresh corn seedling root → A red-yellowish color was perceived that indicates the the presence of reducing sugar. Benedict’s test of 2% sucrose solution without corn seedling root (control) → A blue color was perceived that indicates the absence of reducing sugar. B. Oxireductases a. Dehydrogenase Color intensity of methylene blue After incubation Blue After Aeration With fresh mungbean seedlings initially, the solution appeared to be white and then became blue again yet seemed to be lighter after the performed incubation Without mungbean seedlings (control) Blue Blue b. Catalase Potato tuber strips in 3% H2O2 Appearance of large bubbles in large amounts. Boiled potato tuber strips in 3% H2O2 → Appearance of gas bubbles in small amounts. C. Factors affecting enzyme activity a. Enzyme concentration Color Intensity of Starch –Iodine Solutions With undiluted amylase With half diluted amylase With ¼ diluted amylase After 45 minutes of incubation dark color with light yellowish tinge darker color with yellowish tinge darkest color with brownish tinge b. Hydrogen-ion concentration (pH) Color Intensity of Starch-Iodine Solutions + Amylase (scale of 1 to 7, dark to light) pH 4 buffer 2 (purplish black) pH 5 buffer − pH 6 buffer − pH 7 buffer 4 (bluish green) pH 8 buffer − pH 9 buffer − pH 10 buffer 6 (light blue) c. Temperature Color intensity of Starch –iodine solutions After 45 minutes of incubation At 5-10 ̊C darkest lavender coloration At 28-30˚ C lavender coloration At 98-10 ˚ C lightest lavender coloration Study Questions 1. Give (i) the name of the enzymes catalyzing the following chemical reactions, (ii) their cellular localization, and (iii) the plant physiological process they are involved a) Pyruvate + NAD+ + CoA ----------- acetyl- CoA + NADH + H+ + CO₂ (i) Pyruvate dehydrogenase (ii) Mitochondrion (iii) Citric acid cycle b) Ribulose- 1,5- bisphosphate + CO2 -------------- 2 (3-phosphoglyceric acid) c) 2. (i) Ribulose-1,5- bisphosphate carboxylase (ii) Chloroplast (ii) Carbon fixation Fructose-6-phosphate + ATP ---------------- Fructose-1,6-biphosphate (i) Phosphofructokinase (ii) Chloroplast (iii) Glycolysis Describe the rate of enzyme-catalyzed reaction with increasing substrate concentration. → The rate of reaction catalyzed by an enzyme increases linearly with the substrate concentration up to a point, but it soon reaches the maximum value called Vmax beyond which there is no further increase in reaction rate; this is called substrate saturation. This is so because all of the active sites would be occupied by each substrate. 3. In what ways does hydrogen concentration affect enzyme activity? →In this case, at pH 7, the enzyme activity would be at its optimum level which at this point, the reaction would be at its best activeness. But then, when a pH is reached at this case 10 the, this would cause the denaturation of the proteins yielding a light coloration. And this would mean that the stach-iodine solution changed its structural configuration due to the extreme pH.