This document describes an experiment testing the activity of the ADH enzyme with different substrates and conditions, measuring absorbance to determine enzyme selectivity and efficiency.
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[...] Then, we put 0.05 mL of our ADH enzyme in the even tubes and 0.05 mL of water in the odd tubes (the odd tubes will therefore be the blanks). Then, we incubate all tubes for 5 minutes at 30 degrees, then we add 1 mL of saturated urea at 75 degrees, and we read the absorbance at 340 nm using a spectrophotometer cuvette. Let's analyze our results: We see that the absorbance seems to be increasing (from tube 2 to tube with the concentration of ethanol. [...]
[...] Acetaldehyde + NADH,H+ The ADH is an enzyme present in several organisms, it therefore allows the degradation of alcohol. In humans, ADH is located in the stomach and liver and it allows to catalyze the conversion of ethanol to acetaldehyde : CH3CH2OH + NAD+ ? CH3CHO + NADH + There are also bacterial alcohol dehydrogenases, during this TP, we will study the ADH of a yeast: Saccharomyces cerevisiae. This ADH enzyme is a tetramer, meaning it is composed of 4 perfectly identical subunits. [...]
[...] (We can use the same logic with the tubes without orthophenanthroline c and e). 5. Thermodenaturation of ADH 5.1/- Absence of substrates: Tubes 1 min) 2 (15 min) 3 (30 min) 4 (60 min) Absorbance at 340nm 0.082 0.001 plus les 4 blancs 5.2/- Presence of substrates: Tubes 1 2 3 4 Absorbance at 340nm 0.083 0.001 plus the 4 whites Tubes 1 2 3 4 Absorbance at 340nm 0.122 0.024 0.020 0.029 plus the 4 whites PROTOCOL/ ANALYSIS: The objective of this part is to determine if there is an effect of the substrates on the stability of the enzyme through the denaturation of the ADH enzyme. [...]
[...] We observed that this molecule blocked the enzymatic reaction because it would cause a problem in the tertiary structure of ADH. This experiment shows that, when an enzyme is not in its optimal structure, it will function less well, and the reaction will be less efficient. In the fifth part, we denatured the enzyme thermodynamically. Heated to 75 °C, ADH is denatured, meaning it changes conformation, as a result its affinity with the substrate decreases or becomes null and the reaction cannot be achieved. [...]
[...] Thus, the enzyme would have specificity for certain substrates: it would recognize better 2-Propanol and 1-Propanol than 1-Butanol. We can see that these structures do not really resemble Ethanol, so we can assume that ADH is not a strictly specific enzyme, it is capable of recognizing, in its active site, other molecules than Ethanol. But ADH is not capable of recognizing all alcohols, because, as we saw for tube it is unable to recognize methanol. This enzyme therefore has specificity for alcohols but this specificity has a limit. [...]
