Overview Of Enzyme Kinetics And Inhibition Biology Essay

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ABSTRACT

Polyphenol oxidase is responsible for the browning of fruits and vegetables exposed in the oxygen. Polyphenol oxidase is an example of a tyrosinase which controls the rate of production of melanin, as for the potato, quinone was the product. The Lineweaver-Burke equation was used to determine the rate of the reaction of the inhibition of the enzyme more clearly.

INTRODUCTION

Polyphenol oxidases (PPOs) are a group of copper-proteins, widely distributed phylogenetically from bacteria to mammals that catalyze the oxidation of phenolics to quinones which produce brown pigments in wounded tissues (2). These enzymes are found in the chloroplasts of plants and they could also be released during ripening, handling, storage and processing of fruits and vegetables. They contain copper that oxidizes the phenolic group of a compound to form quinones. Quinones on the other hand, are reactive oxygen molecules bound to a carbon atom by two double bonds. This problem is of considerable importance to the food industry as it affects the nutritional quality and appearance, reduces the consumer's acceptability and therefore causes significant economic impact, both to food producers and to food processing industry(3).

In enzyme kinetics, the Lineweaver-Burke equation and Michaelis-Menten equation were used to determine the rate of the reaction of the enzyme, but for this specific experiment, the Lineweaver-Burke equation is used for a more vivid display of the reaction of the enzyme. The Lineweave-Burke equation can be written as:

This equation is a double reciprocal of the Michaelis-Menten equation that is written as:

The Michaelis constant represented as KM is the reaction rate and the rate of the ES complex formation. This equation presents a hyperbolic curve wherein Vmax is an asymptote of the curve and KM is the substrate.

MATERIALS AND METHODS

The prepared phosphate buffer was cooled while a potato was peeled, grated. The potato was then homogenized along with 40.0 ml of the phosphate buffer and 10.0 ml of 0.100 M NaF for 30 seconds. The homogenate was then filtered and the extract collected was covered with aluminum foil and cooled. To determine the enzyme kinetics of the uninhibited reaction, the following solutions were prepared for analysis shown in table 1.

Table 1. Solution Preparation for the Uninhibited Reaction

Test Tube

0.100 M Catechol

0.200 M Phosphae buffer

1

0.20 ml

2.0 ml

2

0.40 ml

2.4 ml

3

0.80 ml

2.0 ml

4

1.60 ml

1.20 ml

5

2.00 ml

0.80 ml

20.0 ml of the extracted enzyme was added to each test tube. The absorbance of each test tube was read at 420 nm at 5 seconds interval against phosphate buffer using a UV-VIS spectrophotometer. The same amount of the potato extract was added along with 0.100 M Ascorbic acid. Table 2 shows the amount of solutions needed to analyze ascorbic acid. The same procedure was executed for the above data for the Ascorbic Acid.

Table 2. Solution Preparation for the Analysis of Ascorbic Acid

Test tube

0.100 M Catechol

0.100 M Ascorbic acid

0.200 M Phosphate buffer

1

0.20 ml

0.20 ml

2.40 ml

2

0.40 ml

0.20 ml

2.20 ml

3

0.80 ml

0.20 ml

1.80 ml

4

1.60 ml

0.20 ml

1.00 ml

5

2.00 ml

0.20 ml

0.60 ml

RESULTS AND DISCUSSION

Reaction Velocity Determination

Uninhibited Reaction

Table 3. Summary of data of Uninhibited Reaction

Time (s)

Absorbance at 420 nm

Tube 1

Tube 2

Tube 3

Tube 4

0

0.611

0.676

0.763

1.022

5

0.818

0.791

0.859

1.084

10

0.818

0.850

0.929

1.133

15

0.942

0.970

1.004

1.185

20

0.966

0.950

1.004

1.199

25

1.000

0.979

1.028

1.205

30

1.021

1.004

1.044

1.206

35

1.038

1.024

1.052

1.202

40

1.052

0.039

1.056

1.114

45

1.063

1.050

1.058

 

50

1.071

1.059

1.058

 

55

1.075

1.065

1.054

 

60

1.080

1.069

 

 

As time goes, the absorbance of the uninhibited reaction increases and the values are close to each other. Figure 1 shows the slope of the reaction made. It presents an increase in the linear progression of the reaction.

Figure 1. Graph of Uninhibited Reaction

Ascorbic Acid Inhibited Reaction

Table 4. Summary of Inhibited Reaction of Ascorbic Acid

Time (s)

Absorbance at 420 nm

Tube 1

Tube 2

Tube 3

Tube 4

0

0.087

0.125

0.104

0.309

5

0.087

0.123

0.168

0.306

10

0.086

0.122

0.168

0.305

15

0.086

0.122

0.168

0.302

20

0.086

0.121

0.168

0.301

25

0.085

0.121

0.168

0.301

30

0.085

0.121

0.168

0.300

35

0.085

0.121

0.168

0.300

40

0.085

0.120

0.168

0.301

45

0.085

0.120

0.168

0.300

50

0.085

0.120

0.168

0.301

55

0.085

0.120

0.167

0.301

60

0.085

0.120

0.167

0.301

As for the absorbance of the Ascorbic Acid as time goes, the absorbance decreases. There is a slow decrease of the absorbance, making a slow rate of inhibition. The ascorbic acid inhibits the reaction to react rapidly.

Figure 2. Graph of Inhibited Reaction of Ascorbic Acid

The graph shows almost a horizontal line across the others. No slope was formed. The values were quite similar to each other due to the inhibition of the ascorbic acid making no slope at all, leaving a flat diagram.

Since the Lineweaver-Burke equation was used, it demands a velocity. One way to determine the velocity of the reaction was to get the value of the substrate first using the equation:

M1V1=M2V2

From the given formula, S is derived giving off the equation:

TEST TUBE 1

M1= 0.100 M Catechol

V1= 0.20 mL (volume of sample upon addition of Catechol)

V2= 2.6 mL (volume of buffer)

TEST TUBE 2

M1= 0.100 M Catechol

V1= 0.40 mL (volume of sample upon addition of Catechol)

V2= 2.4 mL (volume of buffer)

TEST TUBE 3

M1= 0.100 M Catechol

V1= 0.80 mL (volume of sample upon addition of Catechol)

V2= 2.0 mL (volume of buffer)

TEST TUBE 4

M1= 0.100 M Catechol

V1= 1.60 mL (volume of sample upon addition of Catechol)

V2= 1.20 mL (volume of buffer)

TEST TUBE 5

M1= 0.100 M Catechol

V1= 2.00 mL (volume of sample upon addition of Catechol)

V2= 0.80 mL (volume of buffer)

Figure 3. Computation for the Substrate of each test tube

Table 5. Reaction System of Ascorbic Acid

Reaction System

Velocity (mM/s)

Tube 1

Tube 2

Tube 3

Tube 4

Uninhibited

28

60

173

140

Inhibited

11.63

8.13

5.99

3.28

Lineweaver- Burke Analysis

Table 6. Analysis of the Reaction

Test tube

1/[S], ()

1/V, (s/mM)

Uninhibited Reaction

Ascorbic Acid Inhibited

1

125

0.036

0.086

2

58

0.166

0.123

3

25

0.005

0.167

4

8

0.007

0.305

5

4

0.001

0.352

Figure 4. Lineweaver-Burke Plot

Table 7. Parameters of the Reaction

Parameters

Uninhibited

Ascorbic Acid Inhibited

Vmax

9.01

0.007

Km

0.20

10.17

Mode of inhibition: Competitive Inhibition

When catechol is oxidized it forms benzoquinone. Catechol oxidizes the phenol ring of the aromatic compound with half of the oxygen resulting to benzoquinone and water as the product of the reaction. A quinone is a byproduct of the reaction of the oxidase that results to the browning of the potato upon slicing and other processes.

Figure 5. Structure of Oxidation of Catechol to Benzoquinone

Figure 6. Structure of Ascorbic Acid

The involvement of an inhibitor prevents the browning of the potato. The ascorbic acid reduces the quininone formed by the polyphenol oxidase, PPO, to phenols by oxidation.

Ascorbic acid, also known as Vitamin C, is a water-soluble coenzyme that is found in various types of vegetables and fruits such as lemons, oranges, limes and the like. Ascorbic acid is an effective antioxidant, acting as a free radical scavenger, and an essential cofactor in numerous enzymatic reactions (16). A dietary deficiency of vitamin C can lead to clinical abnormalities such as scurvy, delayed wound healing, bone and connective tissue disorders and vasomotor instability (16). The liver is an important target for the antioxidant effects of vitamin C and plays a role in vitamin body homeostasis, yet the ascorbic acid uptake mechanism in the liver has not been investigated thoroughly (6, 7).