EXPERIMENT ON ENZYME

UMI ABIBAH BT SULAIMAN D20091034811SITI RAHAYU BT MOHAMED NOOR

D20091034855AZMA AMIRA BT MOHAMAD D20091034859

NUR AFIQAH BT MUHAMAD APANDI D20091034872

AMEERA BT YAHYA D20091034814

DR ROSMILAH MISNAN

Abstract The main objective of this study was to prepare standard reference

graph for enzyme reaction concentration and to investigate the effect of substrate concentration, temperature and pH on enzyme concentration. The value of maximum rate of reaction, Vmax and Michaelis constant, Km are obtain from Lineweaver- Burke plot.

From the experiment, Low value of Km means high affinity of the enzyme for the substrate. Increasing the temperature of a system will

increase the number of collisions of enzyme and substrate per unit time. Extremely high or low pH values generally result in complete loss of activity for most enzymes. Amount of hydrogen ion which is related to pH not give effect on the ionic bond of enzyme stucture

and the amino acid sequence of active site in optimum pH. Changed the pH will breaking down the ionic bonding that hold the structure

of enzyme and the charge within amino acid and lead to the failure in formation of enzyme-substrate complexes.

Introduction An enzyme is a biological catalyst that accelerates a

chemical reaction by lowering the energy needed to start the reaction. It causes reactions to happen much faster

than they would if it wasn't present. In our bodies, many thousands of chemical reactions are happening every second. For example, the conversion of sucrose to the

monosaccharide sugars fructose and glucose is a chemical reaction.

METHOD

Part I:

Preparation of standard reference

RESULT

Part I:

Preparation of standard reference

Sta

ndard

gra

phs

METHOD

Part II:

The effect of substrate

concentration

RESULT

Part II:The effect of substrate concentration

Subst

rate

gra

phs

Michaelis- Menten plot (MM)

Discussion

The value of Vmax and Km from Lineweaver-Burke:

1/Vmax = 200 -1/Km = -22

Vmax = 0.005 Km = 0.045

Graph of Michaelis-Menten• the graph at first gradually increase and then

constant at certain values.• concentration of the starch have reached the

maximum value of velocity • After this point, increases in substrate

concentration will not increase the velocity.

Lineweaver-Burke• The value of Vmax is 0.005 while the value of

Km is 0.045• A small Km indicates that the enzyme requires

only a small amount of substrate to become saturated.

• When this maximum velocity had been reached, the entire available enzyme has been converted to ES, which is the enzyme substrate complex.

• Low value of Km means high affinity of the enzyme for the substrate. This is because Km is refers to how well substrate binds to the enzyme.

METHOD

Part III:

The effect of temperature

RESULT

Part III:

The effect of temperature

1/ V

PART 3 : THE EFFECT OF TEMPERATURE

i) At temperature 8 ˚ C

1/[S]

0

200

400

600

800

1000

1200

1400

2.17 2.17 2.17 3.57 11.11 28.57 83.33 125

ii) At temperature28 ˚ C1/ V

1/[S]

0

500

1000

1500

2000

2500

2.13 2.22 5.26 3.57 11.11 30.3 100 200

ii) At temperature 40 ˚ C1/ V

1/[S]

Lineweaver-Burke graph

DISCUSSION

• Enzymes are proteins that catalyze biochemical reactions. An enzyme interacts with a molecule, generally called a substrate, and turns it into a product. The velocity of the reaction depends on the substrate concentration ([S]). But, at a certain concentration, the velocity achieves the maximum value (Vmax).

• m is a Michaelis constant that is the substrate concentration at which the reaction velocity is half of the maximum value. Km can be calculated from the Lineweaver-Burk plot 1/V = (1/Vmax) + (Km/Vmax) x 1/[S].

• Lineweaver-Burk analysis is one method of linearizing substrate-velocity data so as to determine the kinetic constants Km and Vmax. The units for Vmax are the units that you used when entering Y values on the original data table. The units for Km are the units you used when entering the X values.

• V max for the temperature 8˚ C is 2000, for the temperature 28 ˚ C is 1250 and for the temperature 40 ˚ C is 2000. Km for the temperature 8 ˚ C is 105.00. Then Km for temperature 28˚ C is 62.00 and Km for temperature 40˚ C is 100.00.

METHOD

Part IV:

The effect of pH

RESULT

Part IV:The effect of pH

Discussion • From the result, the highest velocity detected at pH 4, 5 and 7. This pH

value gives maximum velocity which provided the suitable environment for the enzyme reaction.

• From the result, this range is optimum pH for amylase, which the enzyme is catalysing the reaction with the maximum reaction. The amount of hydrogen ion which is related to pH not give effect on the ionic bond of enzyme stucture and the amino acid sequence in active site. This range make the enzyme is actively bind to substate and form enzyme-substrate complexes. At the optimum pH, the maximum rate of reaction can be achieved.

• The range for suitable pH in this experiment is 4 to 7 pH, but the reading show the decrease velocity at pH 6. Maybe, this is due to measurement error in handling pipette or absorbance reading

 

• From the theory, the optimum pH for amylase is 6.7 to 7.0 for the pancreatic and saliva amylase (alpha-amylase). For malt amylase (beta-amylase), the optimum pH is 4.6 to 5.2. The gamma amylase need pH 3 for their maximum reaction. For amylase , the difference pH needed for the difference type of amylase enzyme.

• The lowest velocity is at pH 9. At this pH, the amount of the hydrogen ion is decreased, so this amount will breaking the ionic bond of the enzyme structure and the charged in amino acid within the active site. So, the active site is no longer fit to substrate.

 • Changed the pH will breaking down the ionic bonding that hold

the structure of enzyme. So, it lose it functional shape mainly the shape of active site. Consequently, the substrate will no longer fit to active site. The enzyme said to be denatured. The amino acid within the active site will not able to form the enzyme-substrate.

Conclusion

From this experiment,we are able to prepare the standard reference graph for enzyme reaction concentration.We also determine the effect of

substrate concentration,temperature and pH on the enzyme reaction.

References

• Investigating the effect of enzyme concentration on the hydrolysis of starch with amylase

(2011). Get on November 29, 2011 from http://www.123helpme.com/view.asp?id=149093

 • Tor Siong Hoo, Bah Hock Guan, Sri Nasariya (2008).

Matriculation biology. Kuala Lumpur:

Oriental Academic Publication.• Mary K. Campbell (1999). Biochemistry third edition.

United States of America: Saunders and Harcourt College.

APPENDIX

Thank you =)