Enzyme Kinetics And Inhibition Biology Essay

In this experiment, catechol is used as a substrate for the dynamicss of enzyme polyphenol oxidase that was obtained from murphies. A 15.0 g sample of murphy was processed for extraction of polyphenol oxidase and the extracted enzyme was used for finding of rate of chemical reaction in an uninhibited reaction utilizing merely the substrate catechol and the phosphate buffer, and in inhibited reaction, adding ascorbic acid to the reagents. The resulted maximal speed for the uninhibited reaction is 4.37 mM/min while the maximal speed for inhibited reaction is 7.26 mM/min. The Lineweaver-Burke graph plotted utilizing the reciprocal of the substrate concentration and speed shows that the inhibited reaction has an uncompetitive inhibitor.

Introduction

Enzymes are biological, ball-shaped molecules that catalyze or speed up a certain reaction. They cause a specific chemical alteration in all organic structure parts, one illustration is the interrupting down of the nutrient we eat ( 1, 2 ) . Enzymes are made up of amino acids, and these are proteins. Every enzyme has its ain specific function and reaction to rush up depending on its belongings ( 3 ) . Enzyme Kinetics is the procedure wherein the rate of chemical reaction catalysed by an enzyme is measured.

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E + P

Einsteinium

E + S

Figure 1. General Equation for Enzyme-Catalyzed Chemical reaction

As seen in Figure 1, a substrate ( S ) binds to an enzyme ( E ) doing up the ES composite. Afterwards, dissociation of ES composite to the merchandise ( P ) and recycled enzyme ( E ) happens.

MATERIALS AND METHODS

The experiment consists of 3 parts. First is the isolation of Polyphenol Oxidase wherein a 15.0g sample of murphy was peeled and finely chopped. After homogenising it with phosphate buffer in a liquidizer, the infusion was filtered utilizing cheesecloth and was collected in a beaker. Second portion is the Enzyme Kinetics of Uninhibited Reaction wherein 5 trial tubings were prepared with assigned volumes of catechol and phosphate buffer. 0.20 milliliter of the extracted enzyme was so added in every trial tubing before instantly reading the optical density at 420 nm every 5 seconds interval. For the last portion, Enzyme Inhibition, 5 trial tubings same as the 2nd portion was prepared but this case, ascorbic acid was added and the same process for every trial tubing was done. For a more elaborate process, refer to Figure 2.

Isolation of Polyphenol Oxidase

Cover w/ aluminium foil

Phosphate Buffer

Ice bath

+ 40 ml phosphate buffer

Ice bath

Filter

15 g murphy peeled and chopped

Enzyme Kinetics of Uninhibited Reaction

Test Tube

0.100M Catechol

0.200M Phosphate Buffer

1

0.20 milliliter

2.6 milliliter

2

0.40 milliliter

2.4 milliliter

3

0.80 milliliter

2.0 milliliter

4

1.60 milliliter

1.20 milliliter

5

2.00 milliliter

0.60 milliliter

Table 1. Designated sum of Catechol and Phosphate buffer per trial tubing

*Read optical density at 420 nm every 5 sec

Tube 1

* Repeat for every trial tubing

Enzyme Inhibition

Test Tube

0.100M Catechol

0.100M Ascorbic acid

0.200M Phosphate Buffer

1

0.20 milliliter

0.10 milliliter

2.5 milliliter

2

0.40 milliliter

0.10 milliliter

2.3 milliliter

3

0.80 milliliter

0.10 milliliter

1.9 milliliter

4

1.60 milliliter

0.10 milliliter

1.10 milliliter

5

2.00 milliliter

0.10 milliliter

0.70 milliliter

Table 2. Designated sum of reagents per trial tubing

* Repeat process in Part B

Figure 2. Conventional Diagram of Procedure

RESULTS AND DISCUSSIONS

The tabulated informations and consequences of the optical density at 420 nanometer for the Uninhibited and Inhibited reaction can be seen in Table 3 and 4.

Time ( s )

Optical density at 420 nanometers

Tube 1

Tube 2

Tube 3

Tube 4

0

0.746

0.676

0.700

0.480

5

0.773

0.731

0.788

0.795

10

0.798

0.772

0.845

0.867

15

0.820

0.806

0.889

0.914

20

0.837

0.828

0.923

0.955

25

0.861

0.843

0.946

0.986

30

0.877

0.854

0.959

1.012

35

0.877

0.861

0.964

1.032

40

0.899

0.866

0.962

1.044

45

0.910

0.868

0.957

1.049

50

0.912

0.868

0.947

1.042

55

0.899

0.867

0.937

1.029

60

0.870

0.864

0.926

1.011

Table 3. Optical density at 420 nanometer for Uninhibited Reaction

In Table 3, the trial for uninhibited reaction of the enzyme polyphenol oxidase was observed by tabling the reading every 5 2nd interval.

Figure 3. Graph of optical density for Uninhibited reaction

In Figure 3, the graphed signifier of informations tabulated in Table 3 from clip 10 up to 35, or the point wherein the lines ascend were plotted. Every line has its ain designated equation bearing the incline and its y-intercept. The incline of every equation stands for the speed of every reaction in every trial tubing.

Time ( s )

Optical density at 420 nanometers

Tube 1

Tube 2

Tube 3

Tube 4

0

0.23

0.224

0.28

0.341

5

0.23

0.224

0.279

0.34

10

0.231

0.229

0.278

0.34

15

0.26

0.26

0.279

0.34

20

0.304

0.29

0.28

0.341

25

0.347

0.302

0.283

0.343

30

0.385

0.336

0.311

0.346

35

0.417

0.393

0.356

0.424

40

0.443

0.421

0.397

0.472

45

0.462

0.43

0.433

0.509

50

0.474

0.452

0.461

0.546

55

0.485

0.482

0.481

0.573

60

0.496

0.497

0.499

0.602

Table 4. Optical density at 420 nanometer for Inhibited Reaction

Merely like in Table 3, the reading every 5 2nd interval was tabulated for every trial tubing.

Figure 4. Graph of optical density for Inhibited Reaction

Chemical reaction System

Velocity ( mM/s )

Tube 1

Tube 2

Tube 3

Tube 4

Uninhibited

3.4

3.5

4.7

6.6

Inhibited

5.5

5.6

6.5

8.1

Table 5. Speed of enzyme in Uninhibited and Inhibited reaction

The speed tabulated in Table 5 was obtained by looking at the incline of every equation in the graphs plotted. Every incline value was multiplied by 1000 to change over it to mM unit.

Tube #

1/ [ S ] , ( mM-1 )

1/V, ( s/mM )

Uninhibited Chemical reaction

Ascorbic Acid inhibited

1

0.1499

0.294

0.182

2

0.075

0.286

0.179

3

0.0375

0.213

0.154

4

0.0188

0.152

0.123

5

0.015

0.333

0.152

Table 6. Lineweaver-Burke Analysis

The reciprocal of the substrate concentration computed by utilizing the dilution expression M1V1 = M2V2 was tabulated in Table 6 together with the reciprocal of the speed both in uninhibited and inhibited reaction.

Figure 5. Lineweaver-Burke Graph

Parameters

Uninhibited

Ascorbic Acid inhibited

Vmax

4.37

7.26

KM

1.97

2.48

Mode of Inhibition: Uncompetitive Inhibition

The graph in Figure 5 shows that the two lines are parallel with each other showing an uncompetitive suppression.

Figure 6. Oxidation of Catechol to Benzoquinone

Figure 7. Structure of Ascorbic acid

Figure 8. Mechanism of Ascorbic Acid as an antioxidant