FUN EXPERIMENT 3 : Enzyme

INTRODUCTION

 Enzymes are the catalysts of biological system and are extremely efficient and specific as a catalyst. Compared the acceleration the rate of reaction by factors with the absent of enzyme, a reaction can be speed up at least a million times with the presence of enzyme with the same reactions. Enzymes are highly specific and only particular enzyme catalyses only a single chemical reaction or a set of closely related chemical reactions.

Enzyme kinetics is study of the rates of enzyme-catalysed reactions. It’s principally concerned with the measurement and mathematical description of this reaction rate and its associated constants. To investigate the enzyme rates, Michaelis-Menten equation (MM) is used. In this experiment, in order to discuss properties of an enzyme (amylase), certain values are determined experimentally under steady state conditions. These values are determined by using formula for kinetics studies that include;

Vmax : The so-called maximal rate of the catalysed reaction. The enzyme's active site is    

           saturated.

Km : The Michaelis constant. The substrate concentration at which the reaction rate is one 
          half of its maximum value or also known as the turn over number. 

These values are determined experimentally by recording the progress of an enzyme-catalysed reaction using fixed amount of enzyme and a series of different factors such as temperature, pH and substrate concentration are test. These values used to construct a graph that presenting the reaction rate occur. Different graph assemble different reaction occur such as enzyme inhibition that act in varies of ways that affect their kinetic effect.

ABSTRACT

The purpose of this experiment conduct was to determine the effect of substrate concentration, temperature and pH on enzyme activity. In this experiment we want to evaluate absorbance and the reaction rate of enzyme amylase in starch-iodine solution. The enzyme activity was measured through its absorption rate in the spectrophotometer with a wavelenght of 590 nm. To measure the result of each effects, we had plotted one standard reference graph from a 9 standard solution of starch (0, 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7 and 1.0 mg/ml) as a So. To test the effect of substrate concentration in enzyme activity, each standard solution was prepared in different concentration of water and amylase. Then, through the testing the enzyme at different temperatures and pH levels, it would determine at which temperature and pH level the enzyme worked the most efficiently. To test the optimal pH, the starch and a buffer were combined at a specific pH level and each sample was incubated for 10 minutes. For temperature, we used three different temperature of (8, 28, 40ºC) to determine the rate reaction of enzyme. Then, the solution was tested in spectrophotometer of 590 nm. Specific graphs were drawn to determine at which substrate concentration, pH and temperature was the optimum for the enzyme after the experiment. Through our result that we obtained,  the enzyme amylase worked more efficiently under the pH level of 6 and 7.  Michaelis- Menten kinetic theory proved that there is the relationship of substrate concentration to the reaction of the velocity.The lower the value of Km, the higher the affinity of the enzyme for enzyme substrate complex formation. For temperature, Enzyme reacts optimally at V_max  0.01 where the affinity of the substrate is higher while the K_m  is lower, 0.005. However, since the whole testing experimental was taken only once, we cannot state that the results were precisely accurate

MATERIALS:

• Test tube
• Amylase
• Iodine
• Distilled water
• Waterbath
• Ice
• Spectrophotometer

PROCEDURES:

          A)   Preparation of standard reference

•     Starch solutions were prepared from the stock solution (1.0 mg/ml) into dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution.
•      Iodine solution is prepared by adding 5 g potassium iodide to 100 ml water. The dissolved potassium iodide is added with 1 g of iodine and is allowed to dissolve.
•      A standard curve of Absorbance (@ 590 nm) vs Concentration of a starch/iodine mixture was prepared. 

      B)   The effect of substrate concentration.

•     Standard starch solution was prepared into dilutions of 0.01, 0.025, 0.05,0.1, 0.3,0.5,0.7 and 1.0 mg/ml from the starch stock solution.
•     The starch hydrolysis in different substrate concentration was prepared as the table below.

• Starch concentration was calculated after each sample undergoes hydrolysis by using standard curve. Starch concentration was calculated by using initial starch concentration and final starch concentration, [S]= (S_o) – (S_f)
• The velocity was calculated as V= ∆S/∆ t = (S₀ – S_f) / 10 minutes
• By using the data collected, a Michaelis-Menten graph was plotted.
• The value of V_max and Michelis constant K_m were stated.

          C)   The Effect of Temperature  

•      The following table below is uses as guide in an experiment the effect of temperature on enzyme.
• 
•     The Lineweaver-Burke line was plotted for the result of 20, 28, 35 and 40°C.
•     All three plots in Lineweaver-Burke line were being compared.
•     In the Lineweaver-Burke line, the values of Vmax and Km for all plots were find.

       D)   The effect of pH

•     The following table below is uses as guide in an experiment the effect of pH on enzyme

•     The values of V for all pH were stated.
•     The velocity for each pH test was compared. 

   

         RESULT AND DISCUSSION:

  

         A) Standard Graph 

B) The effect of Substrate Concentration

·  Michaelis-Menten graph is a graph that shows the relationship between the concentrations of a substrate and the rate of the corresponding enzyme controlled reaction. Michaelis-Menten kinetic theory of enzyme action state that enzyme concentration and substrate concentration give effect on the velocity reaction. The effect of enzyme concentration on reaction velocity means that if the substrate concentration is held constant, the velocity of the reaction is proportional to the enzyme concentration.

·      In this experiment, we are focusing on the second kinetic theory of enzyme action which is the effect of substrate concentration on reaction velocity. There are two orders that present in the Michaelis-Menten graph which are first order and zero order. When the substrate concentration is low, the reaction velocity is first order with respect to the substrate means that the reaction of velocity is proportional to the substrate concentration. But, when the substrate concentration becomes higher, the reaction velocity is zero order means that velocity is independent of substrate concentration. At middle of substrate concentration, the proportional of the reaction is changing.

·         There are two parameters that involve in the Michaelis-Menten graph which is Vmax and Km. Vmax is the maximum rate at which the enzyme can catalyze the reaction while Km or Micahelis constant is the substrate concentration at half Vmax. From the results, the maximum velocity is 0.062 and half Vmax is 0.031. Then, Km was obtained from the graph at half Vmax and the value is 0.24. The Michaelis constant,Km provide a measure of the affinity of an enzyme for its substrate. The lower the value of Km, the higher the affinity of the enzyme for enzyme substrate complex formation.

C) The effect of temperature

•    For this experiment, we study about the relationship of activity of enzyme and temperature. Before explaining the graph, we should explain to you about the V_max and K_m that related to the enzyme activity calculations.  V_max refers to the velocity maximum that the enzyme can reach when certain temperature apply to a reaction, whereas, K_m  is Michealis constant, which shows us the affinity of an enzyme in a reaction. The lower K_m  readings the higher affinity of the enzyme

•    In the calculation above, we can see that V_max for temperature is 0.05. In a reaction the higher velocity the higher rate of reaction. This is because more collisions occur in between enzyme and substrate. As the temperature rises, molecular motion and hence collisions between enzyme and substrate speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective. 
• K_m for 8^oC is 0.42 , for 28^oC is 0.4 whereas, for 40^oC is 0.3. The type of inhibitor of the reaction is known as competitive inhibitor.
•  The K_m value is lower indicates that the enzyme have the higher affinity to react.

    

            D) The Effect of pH

·  Based on the result obtained in Table 4, we can see that the highest velocity of the enzyme reaction is at pH 6 and pH 7 which is 0.045 V. The velocity increased rapidly from pH 5 to pH 6 at 0.003 V.

·    Based on Table 4, it shows that amylase used in this experiment is highly active at pH 6 to pH 7. This can be shown by the static amount of velocity at both pH values. The velocity decreased rapidly from pH 8 to pH 10. This shows that the enzyme cannot react in those pHs. Thus, this shows that the enzyme can react actively at pH below 7.

·  In the theory, it was stated that there are three types of amylase enzyme. The α-Amylase, β-Amylase, γ-Amylase enzyme.

·    Each types of amylase active optimally at different pH.  The α-Amylase react optimally at pH 6.7 to 7. The optimum pH for , β-Amylase is pH 4.0 to 5.0.  While γ-Amylase optimally at pH 3.

·  From the result shown in Table 4, the enzyme reacts actively at pH 6 and pH 7. Therefore, it is predicted that alpha amylase was used in this experiment.

·     While conducting the experiment, there are few error causes us failed to obtained the exact result. One of the major errors occurs is the using of dropper instead of micropipette. The function of the micropipette is to measure a small reading of volume needed. In this experiment, the smallest volume used is 1 mL. We used the dropper to measure and sometimes parallax error might occur during the measurements thus giving the wrong reading for the volumes. Error in volume of concentration might give the error for the absorbance reading. Thus it might affect the result for the enzyme activity.

·   During taken the measurement for the pH, we also used the same of dropper and measuring cylinder. We might rinse both of dropper and micropipette with not proper ways. This might contaminate the others pH value. 

CONCLUSION:

From these enzyme experiment, we can conclude that,

•   In a nutshell, Michaelis- Menten kinetic theory proved that there is the relationship of substrate concentration to the reaction of the velocity.
•       Enzyme reacts optimally at V_max is 0.01. The affinity of the substrate is higher as the K_m  is lower which is 0.005.
•      The enzyme, amylase reacts actively at pH 6 and pH 7.

REFERENCES:

·     Donald B.Sittman, Victor L. Davidson (1999). BIOCHEMISTRY 4^TH EDITION.  Philadelphia : Lippincott-Raven Publisher.

·         http://en.wikipedia.org/wiki/Enzyme

·         http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html

·         https://en.wikipedia.org/wiki/Velocity

·         http://en.wikipedia.org/wiki/Amylase