Exercise 6

ENZYMES

Results and Discussions

Name: Ma Chrischelle A. Ferreras Section: HJK Date Performed: Dec. 4-5, 2009

Group name: Two is Better than One Group (TIBTO) Date Submitted: Dec. 29, 2009

A. Hydrolases

a. Amylase

Iodine test of 0.1% starch solution with corn seedling root after incubation:

dark violet

Iodine test of 0.1% starch solution after incubation:

darker violet

b. Sucrase

Benedict’s test of 2% sucrose solution with fresh corn seedling root:

yellow

Benedict’s test of 2% sucrose solution without corn seedling root (control):

clear
B. Oxireductases

a. Dehydrogenase

Color intensity of methylene blue

after incubation after aeration

With fresh mungo bean seedlings white white
Without mungo bean seedlings (control) clear blue clear blue

b. Catalase
Potato tuber strips in 3% H2O2 gas evolution observed
Boiled potato tuber strips in 3% H2O2 no gas evolution observed

C. Factors affecting enzyme activity

a. Enzyme concentration

Color intensity of starch-iodine solutions

after 45 minutes of incubation

1) With undiluted amylase yellow

2) With half diluted amylase lightest yellow
3) With ¼ diluted amylase pale yellow

b. Hydrogen-ion concentration (pH)

Concentration of starch-iodine solutions + amylase

(scale of 1 to7, dark to light)

pH 4 buffer (pink) dark (darkest)

pH 7 buffer (green) light
pH 9 buffer (blue) light
pH 10 buffer (blue) lighter (lightest)

c. Temperature
Color intensity of starch-iodine solutions + amylase
after 45 minutes of incubation

1) At 5-10°C violet (darker)

2) At 28-30°C violet (light)
3) At 98-100°C violet (darkest)
Study Questions

1. Give (i) the name of the enzymes catalyzing the following chemcal reactions, (ii) their
cellular localization, and (iii) the plant physiological process they are involved.

a) Pyruvate + NAD+ + CoA -------------> acetyl-coA + NADH + H+ + CO2

i. Coenzyme A
ii. cytoplasm
iii. glycolysis

b) Ribulose-1,5, bisphosphate + CO2 ------------> 2(3-phosphoglyceric acid)

i. RuBp carboxylase
ii. Stroma of the chloroplast
iii. Calvin cycle

c) Fructose-6-phosphate + ATP ------------> fructose-1,6-biphosphate

i. Phosphofructokinase
ii. Cytoplasm
iii. Glycolysis

2. Describe the rate of enzyme-catalyzed reaction with increasing substrate concentration.

The enzyme is said to be in saturation under conditions of high substrate

concentration, thus its active site is always occupied by substrate or product
molecules. Further increase in substrate concentration will not affect the
reaction rate because all active sites are already in use in this condition.
3. In what ways does hydrogen ion concentration affect enzyme activity?

Most enzymes have an optimum pH at which their activity is

characteristically maximal. Above or below this pH value, enzyme activity,
decline. The protein’s three-dimensional structure is altered and the H +
concentration (pH) in the medium is changed drastically. Extreme changes in
pH can cause denaturation. Acids and bases alter a protein’s three-
dimensional structure because the H+ and OH- compete with hydrogen and
ionic bonds in an enzyme, resulting in the enzyme’s denaturation.