REPORT 2: ENZYME

Title : Enzyme kinetics

Objective : 

To determine the effect of the substrate concentration, temperature and pH on enzyme velocity.

Introduction :

 Enzymes are catalysts which lower the activation of chemical reactions, thus making them happen more rapidly. The behavior or activity of an enzymes can be affected by pH, temperature, and substrate concentration.

Materials :

    Starch solutions

Distilled water

Iodine solutions

Amylase

Test tube

 pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10

Procedure :

1. Preparation of standard reference

a) Starch solutions from the stock solutions (1.0 mg/ml) was prepared into dilutions of 0.1, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg/ml from the starch stock solution.

b)The starch solution was mixed with distilled water and iodine solution. Iodine solution was prepared by added 5 g potassium iodide to 100 ml water.

c) A standard curve of absorbance (@590 nm) vs concentration of starch/iodine mixture was prepared. The following table was used as guide.

Table 1

Preparation of starch/iodine mixture for standard curve determination

a) The absorbance was measured at 590 nm

b)  A standard curve of absorbance (@590 nm) vs concentration of starch/iodine mixture was ploted.

2. Determination the effect of substrate concentration, temperature and pH on enzyme velocity.

A.  The effect of substrate concentration

i. Experiment of starch hydrolysis in different substrate concentration was prepared as the following table.

 

Table 2

B.  The effect of temperature

i.The following was prepared for the experiment of  different temperature.

Table 3

 

C. The effect of pH

i. The following was prepared for the experiment using different pH.

Table 4

Result 

The effect of substrate concentration

Effect of pH

Michaelis-Menten Plot:

V_max = 0.099 mg/ml min

V_max/2 = 0.0495 mg/ml min

K_m = 0.24 mg/ml

Lineweaver-Burke Plot:

1/V_max = 60 (mg/ml min)­­^-1

V_max = 0.017 mg/ml min

-1/ K_m =- 4 (mg/ml)^-1

K_m = 0.25 mg/ml

Discussion

            Maximun rate of reaction, V_max, describe the number of substrate being catalysed per second or minute. At V_max, the enzymes are saturated with substrate. Michaelis constant, K_m describes the affinity or strength of binding of the substrate to the active site of an enzyme. The lower the K_m, the greater the affinity. Therefore, smaller concentration of substrate is needed to achieve V_max. K_m of an enzyme is importance in predict whether or not the rate of formation of product will be affected by the availability of substrate.

            The K_m of an enzyme depends on particular substrate and conditions such as temperature and pH. Michaelis-Menten graph is plotted to determine the effect of substrate concentration to enzymes velocity. The V_max and K_m obtained from the graph is 0.099 mg/ml min and 0.24 mg/ml. However, it is difficult to fit the best hyperbola through experimental points, and difficult to determine V_max with any precision by estimating the limit of hyperbola at infinite substrate concentration.

            Thus, Lineweaver-Burk graph is plotted. The linear relationship permit more precise fitting to the experimental point and estimation of the values of K_m and V_max. The V_max and K_m obtained from the graph is 0.017 mg/ml min and 0.25 mg/ml. The low K_m relative to concentration of substrate show that enzyme will act at constant rate regardless of the variations in the concentration of substrate. Therefore, the starch concentration does not affect the amylase velocity.      

            The Lineweaver-Burk graph is plotted to determine the effect of temperature on enzyme velocity. The V_max for the plots of all temperature is 0.057 mg/ml min. The K_m for 8 , 28 , 60 , and 100  is 0.250 mg/ml, 0.125 mg/ml, 0.083 mg/ml, and 0.0417 mg/ml. At high temperature such as 60 , and 100  , the K_m is very low. This means that the enzymes are saturated with substrate with small amount of substrate. This is because enzymes denatured at high temperature thus only little enzymes available to bind with substrate. Therefore, temperature affect the amylase velocity.   

The pH will affect the enzyme velocity. From pH 4 to pH 7, the velocity of the enzyme increases. The velocity of the enzyme is constant at pH 8 and 9. The velocity of the enzyme starts to decrease at pH 10. This showed that the optimum pH for enzyme amylase is pH 8 and 9.     

Reference:Mary K. Campbell, Shawn O. Farrell (2015) Biochemistry.8th Ed. Cengage Learning

Garret, R.H., & Grisham, C.M. (2013). Biochemistry (3rd ed.). USA: University of Virginia

Nelson, L.D and Cox, M.M. (2012) Principles in Biochemistry (6th Ed). Lehninger. W.H. Freeman. 

Reflection:

1. TING MEE PING (D20141067055)

From this experiment, I have learnt that the enzymes activity will be affect by temperature and pH. The high temperature and pH beyond the optimum pH will denature the enzyme. The substrate concentration does not affect the enzymes activity. The low K_m relative to concentration of substrate show that enzyme will act at constant rate regardless of the variations in the concentration of substrate. 

2. CHRIS ROXA ANGELLA SUPAIN (D20141067094)

From this experiment that we had conduct, i have learnt some characteristic of enzymes. Temperature and pH of environment affect the enzymes activity. The higher the temperature, the faster the enzymes activity. However too high of temperature will cause the enzyme to denature and can not perform it function. Same goes with pH value, different enzymes have different optimum pH. Beyond the optimum pH will cause enzyme denaturation. 

3. LENDRA NALAT ( D20141067021)
In this experiment, I have learned that enzymes are biocatalysts that increase the rate of chemical reaction by lowering the activation energy of the reaction. Enzyme participate in the reaction but remain unchanged at the end of the reaction. Through this experiment, i also learned that enzyme activity can be affected by several factors such as pH, temperature and substrate concentration.