Enzymes are important substances made by the cells of plants and animals. There are called catalysts, which are substances that control how quickly chemical reactions occur without interfering at the reaction products (Daja and Treska 2015). Structure of both the enzyme and substrate, the substance on which an enzyme acts; provides clues as to how an anticipated reaction can be favored by the alignment of catalytic residues (Johnson 2008). The reaction of an enzyme is represented as: In this equation, when there is enough substrate (S) to completely alter the enzyme (E) to the enzyme substrate intermediate (ES), the first step in the reaction, the formation of ES is reversible. However, the second step, the formation of the product (P),
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Similarly to the first procedure, readings were taken every 30 seconds after the 15 seconds of mixing, removing the cuvette from the spectrometer between readings. This was repeated for the other five samples. The concentration of catechol, the substrate, was calculated for all six samples and converted to ku so that we could plot ku/min v vs. [S], and to complete a Michaelis-Menten plot of 1/v versus 1/[S] to calculate maximum velocity of the enzyme, as well as determining Km. For part three which was determining the nature of the active site, six additional cuvettes were prepared by adding water, 10 mM catechol, and 50 mM phosphate buffer for a total volume of 1.35ml represented below:
Water (mL) Catechol (mL) Buffer (mL)
0.60 0.0 0.75
0.54 0.06 0.75
0.48 0.12 0.75
0.42 0.18 0.75
0.36 0.24 …show more content…
The mode of inhibition of Cinnamic acid was determined as competitive. This supported my hypothesis because only Km changed while VMax pretty much remained the same. The way Cinnamic Acid interacted with phenoloxidase was it bound to the active site on phenoloxidase, preventing the binding of the substrate (catechol) to occur. This is a result of the structure of Cinnamic Acid closely resembling the chemical structure of catechol, allowing it to be recognized by the enzyme’s active site. It may have still interacted with the enzyme at the active site, but no reaction was able to take place since the inhibitor was stuck on the enzyme, stopping any substrate molecules from reacting with the enzyme (Ophardt
Similarly to the first procedure, readings were taken every 30 seconds after the 15 seconds of mixing, removing the cuvette from the spectrometer between readings. This was repeated for the other five samples. The concentration of catechol, the substrate, was calculated for all six samples and converted to ku so that we could plot ku/min v vs. [S], and to complete a Michaelis-Menten plot of 1/v versus 1/[S] to calculate maximum velocity of the enzyme, as well as determining Km. For part three which was determining the nature of the active site, six additional cuvettes were prepared by adding water, 10 mM catechol, and 50 mM phosphate buffer for a total volume of 1.35ml represented below:
Water (mL) Catechol (mL) Buffer (mL)
0.60 0.0 0.75
0.54 0.06 0.75
0.48 0.12 0.75
0.42 0.18 0.75
0.36 0.24 …show more content…
The mode of inhibition of Cinnamic acid was determined as competitive. This supported my hypothesis because only Km changed while VMax pretty much remained the same. The way Cinnamic Acid interacted with phenoloxidase was it bound to the active site on phenoloxidase, preventing the binding of the substrate (catechol) to occur. This is a result of the structure of Cinnamic Acid closely resembling the chemical structure of catechol, allowing it to be recognized by the enzyme’s active site. It may have still interacted with the enzyme at the active site, but no reaction was able to take place since the inhibitor was stuck on the enzyme, stopping any substrate molecules from reacting with the enzyme (Ophardt