Exercise 6

ENZYMES

Results and Discussion

Name: Ivy Ann C. Carba Section: HJK Date Performed: Dec. 4-5, 2009

Group name: Hale’s Prodigal Daughters 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 Aeration
After incubation

With fresh mungbean seedlings

initially, the solution appeared
to be white and then
became blue again yet seemed
to be lighter after the
Blue
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

After 45 minutes of incubation

With undiluted amylase dark color with light yellowish tinge
With half diluted amylase darker color with yellowish tinge
With ¼ diluted amylase 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)

(i) Ribulose-1,5- bisphosphate carboxylase

(ii) Chloroplast

(ii) Carbon fixation

c) Fructose-6-phosphate + ATP ---------------- Fructose-1,6-biphosphate

(i) Phosphofructokinase

(ii) Chloroplast

(iii) Glycolysis

2. 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.