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Enzymes catalyze biochemical reactions by lowering their activation energy. Most of them are proteins. They are specific to their substrates. Indeed, their specificity is variable; albumin can form complex with many substrate, whereas DNApolymerase is highly specific.
To analyze enzyme kinetics Michaelis-Menten equations are used. This kinetic model is relevant to situations where the concentration of enzyme is much lower than the concentration of substrate, and when the enzyme is not allosteric. The general equation for an enzymatic reaction can be written as below:
Figure 1: Michaelis-Menten Plot 
If the Michaelis-Menten Equation is written as below:
It will become in this lineer equation form:
So the enzyme kinetics can be plotted as a lineer fuction of Km and Vmax.
Figure 2: Lineweaver-Burk Plot 
Competitive Inhibition: The inhibitor binds to the substrate from the active site, thus inhibits the binding of the enzyme to the active site. Inhibitor and the enzyme compete to bind to the active site of the enzyme. The Lineweaver-Burk Competitive Inhibition plot will be as below:
Figure 3: The Lineweaver-Burk Competitive Inhibition plot 
Noncompetitive Inhibition: The inhibitor binds to the substrate not from the active site but from the allosteric site. This causes a conformational change in the substrate, preventing the binding of the enzyme to the substrate, since the active site of the enzyme has changed. The Lineweaver-Burk Noncompetitive Inhibition plot will be as below:
Figure 4: The Lineweaver-Burk Noncompetitive Inhibition plot 
Tyrosinase: This enzyme catalyzes the oxidation of phenol groups. It contains copper. It is responsible of potato blackening and also the disease albinism.
Figure 5: Oxidation of DOPA in the presence of tyrosinase 
Cinmanic Acid is the inhibitor for tyrosinase.
Figure 6: Cinmanic Acid 
MATERIALS & CHEMICALS
L-Dopa (20 mL pergroup, 7.60 x 10-3 M)
Sodium phosphate buffer 0,05 M; pH 7
Cinnamic acid, 50mg/100mL
Mushroom tyrosinase, 100 units/ml
Sodium phosphate buffer, L-DOPA and cinnamic acid solutions are prepared before the experiment.
5 test tubes are prepared. L-DOPA is added 0.75 ml to all test tubes.
Buffer is added to all test tubes at different amounts.
Tyrosinase is added to make the solution 3 ml in total.
Absorbance values are measured in every 30 seconds in the time interval of 2 minutes.
Test tubes are prepared and tyrosinase amount is kept constant at 0.05 ml.
L-DOPA is added differently in all tubes; 0.05ml , 0.1ml , 0.2 ml, 0.4 ml, 0.5ml , 0.75ml.
Buffer amount is added according to the calculations to make the solution 3ml in total.
Absorbance values is measured in every 30 seconds for 2 minutes.
Test tubes are prepared. Tyrosinase is kept constant at 0.05ml and cinnamic acid at 0.4 ml.
L-dopa is added differently in all tubes; 0.05ml, 0.1ml, 0.2ml, 0.4ml, 0.5ml, 0.75ml.
Buffer is added to the tubes to complete the solution to 3 ml in total.
Finally, the absorbance values are measured in every 30 seconds for 2 minutes.
Tyrosinase activity. DOPAchrome formation can be observed in 465 nm wavelength.
Where ÎÂµ=3600M-1cm-1, b=1 cm
Tyrosinase Activity ââ‚¬" 465nm
at 280 nm
at 280 nm
Cinnamic acid (mL)
at 280 nm
Plot 1. Lineweaver-Burk Plot for Tyrosinase Activity
Plot 1. Lineweaver-Burk Plot for competitive Tyrosinase Activity
In this experiment our aim was to learn the mechanism to measure enzyme activity through enzyme mushroom tyrosinase. The enzyme activity shows us the affect of inhibitor. Tyrosinase enzyme is used in this experiment. The kinetics of the three different samples with different amounts of enzymes and buffers are found. In order to understand enzyme kinetics, reaction rates of tyrosinase with and without an inhibitor are measured. The substrate of tyrosinase is L-DOPA, cinmanic acid is the inhibitor for tyrosinase during DOPA oxidation.
The concentration of the solution which is catalyzed by an enzyme is found. The absorbance values are measured. As the time passes, the higher absorbance values can be read. After a while, absorbance becomes constant and as the substrate totally turned int the product.
The color of the solution turned into orange, as the enzyme tyrosinase shows its activity. With greater tyrosinase activity, thus with time the solution color darkened.
In the first part of the experiment tyrosinase acitivity was measured without the presence of any inhibitor. DOPAchrome formation with varying DOPA concentration was measured by determining the solution absorption after every 30 seconds. First the substrate and buffer solutions were added to the vials and then with tyrosinase addition, the measurements start, since enzyme addition initiates the reaction.
DOPA chrome formation was first very quick, whereas after 1.5 minutes the reaction came to an end. This is due to the saturation of the substrate with enzyme. Because all the substrates are bound to enzymes, no more reaction will be possible. The color darkness also indicate this slowing, after some time the orange color did not get darkened any more. Thus color is a direct indicator of the concentration.
In the second part beside varying substrate and same content of enzyme, an inhibitor was added to the solutions. Again the vmax and KM values were determined. The inhibitor is noncompetitive. So, the enzyme activity is inhibited and causes a conformational change. Its binding provides to decreasing the speed of the reaction. The graphs show us that less amount of product is formed in same period of the two different experiments( absence of the substrate and the existence of it)
Theoretically the two enzyme rates should be equal, since the enzyme concentration and other factors, such temperature, pH, pressure were same in both of the cases. The rate should not be effected by the presence of any inhibitor, because Vmax is a function of all enzyme molecules uniting with substrate. The Michaelis constants for two cases should be differing, because obviously it requires larger concentrations of substrate to overcome the competition of the inhibitor for the active site.