To Observe Enzyme Kinetics Biology Essay

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Enzymes are catalysts, usually proteins, that decrease the activation energy needed for the reaction and thus they speed up the rate of a chemical reaction. Every enzyme binds to it substrate specifically, where the enzyme binds to its substrate called the activation site. There are several factors that effect the activity of enzymes; concentration of substrate molecules, temperature, presence of inhibitors and pH. [1] [2]

2.2) Inhibitors

Substances that can reduce or stop the activity of an enzyme by blocking or distorting the active site of the enzyme are called inhibitors. There are two types of inhibitors; competitive or non-competitive. Competitive inhibitors binds to the active site of the enzyme so, they block it for the substrate. They compete with the substrate molecule to bind to the active site. Non-competitive inhibitors binds to other parts of the enzyme and makes a conformational change in the active site of the enzyme so that the substrate can't bind to the active site because there is a lock and key relation between them. [2]

2.3) Michaelis-Menten equation:

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The michaelis menten model gives information about how an enzyme works and the enzyme kinetics. The equation relates the initial reaction rate to the substrate concentration. It describes the rates of irreversible reactions. [3]

E: enzyme

S: substrate

ES: enzyme-substrate complex

P: product

Vmax: maximum velocity achieved by the system at maximum substrate concentration

Km: the Michaelis constant, substrate concentration at which the reaction velocity is the half of the maximum velocity.

Michaelis-menten graph is not linear. It is difficult to estimate Km and Vmax accurately so there are some linerizations developed; Lineweaver-Burke plot, Eadie Hofstea diagram and Hones-woolf plot.

3) EQUIPMENT AND CHEMICALS

3.1) equipments:

Cuvettes

Spectrophotometer

Pipette

Tips

Tubes

3.2) chemicals:

Sodium phosphate buffer 0,05 M; pH 7

Mushroom tyrosinase, 100 units/ml

L-Dopa (20 mL pergroup, 7.60 x 10-3 M)

Cinnamic acid, 50mg/100mL

4) PROCEDURE

Sodium phosphate buffer, L-DOPA and cinnamic acid solutions were prepared before the experiment.

5 test tubes were prepared. L-DOPA was added 0.75 ml to all test tubes.

Buffer was added to all test tubes at different amounts. (buffer amounts were calculculated because we took the half of the solutions that is written and tried to sum the whole solution up to 3 ml.

Tyrosinase was added to make the solution 3 ml in total.

Absorbance values were measured in every 30 seconds in 2 minutes.

Test tubes were prepared and this time tyrosinase amount was kept constant at 0.05 ml.

L-DOPA was added differently in all tubes; 0.05, 0.1, 0.2, 0.4, 0.5, 0.75.

Buffer amount was added according to the calculations to make the solution 3ml in total.

Absorbance values were measured in every 30 seconds for 2 minutes.

Test tubes were prepared. Tyrosinase was kept constant at 0.05 and cinnamic acid was kept constant at 0.4 ml.

L-dopa was added differently in all tubes; 0.05, 0.1, 0.2, 0.4, 0.5, 0.75.

Buffer was added to the tubes to complete the solution to 3 ml in total.

Absorbance values were measured in every 30 seconds for 2 minutes.

5) RESULTS

Sample 1:

30 seconds:

Sample

Absorbance

1

0.860

2

0.890

3

0.940

4

0.940

5

0.940

60 seconds:

Sample

Absorbance

1

0.820

2

0.940

3

0.950

4

0.930

5

0.940

90 seconds:

Sample

Absorbance

1

0.880

2

0.920

3

0.930

4

0.930

5

0.940

120 seconds:

Sample

Absorbance

1

0.890

2

0.930

3

0.940

4

0.950

5

0.970

Sample 2:

120 seconds:

Sample

Absorbance

1

0.700

2

0.930

3

0.940

4

0.950

5

0.950

6

0.945

Sample 3:

120 seconds:

Sample

Absorbance

1

0.205

2

0.345

3

0.500

4

0.790

5

0.820

6

0.890

6) DISCUSSION:

In this experiment our purpose was to observe enzyme kinetics and to observe the changes when an inhibitor binds to the enzyme. We used tyrosinase enzyme in this experiment. And in order to observe the kinetics we prepared three different samples with different amounts of enzymes and buffers. And in one of the samples we used an inhibitor.

In the first sample we took the L-dopa amount the same in all the tubes. And we changed the enzyme amount and buffer amount were changed. The graphs which were obtained in two minutes for every 30 seconds, shows us that after a certain time the rate reaches to its limit. It's the same for the second sample. In this sample we used constant amount of enzyme this time, and changed the others. In the third sample we added an inhibitor. It was a non-competitive inhibitor, cinnamic acid. Which means it decreases the speed of the reaction. It makes a conformational change in the active site of the enzyme. And because of it enzymes interest for the substrate is not changing. When we look at the graphs and compare them with and without the substrate, less amount of product is formed in the same time period.

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