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Enzyme Kinetics - Alkaline Phosphatase


            
             In this lab we studied how the enzyme alkaline phosphatase (AP) activity was influenced by time, the change of temperature and how the enzyme was affected by different substrate and inhibitor concentrations. In this series of experiments alkaline phosphatase will catalyse the reaction where nitrophenyl phosphate (pNPP) is dephosphorylated to form p-nitrophenol and inorganic phosphate. ┬áThe substrate, pNPP, is colourless and the product, p-nitrophenol, has a yellow color. Thanks to this transformation one can easily see if a reaction has taken place and to what extent. .
             We performed a series of 3 experiments and the purpose of these was to test how different variables, such as temperature and inhibitors, affect the activity of the enzyme and therefore the production of the product. The purpose of the first experiment, which was a theoretical one, was to determine the time interval for the initial velocity V0.This experiment was only done theoretically and it determined for how long the reaction can proceed without significant deviation from V0 by using an already existing set of data. .
             We came to the conclusion that 10 minutes was the appropriate time interval and we later used this result as the incubation time in the two following experiments. In experiment 2 we measured the activity of our enzyme AP at six different temperatures, 0, 20, 40, 60, 80 and 100oC. The purpose of this was to find the optimal temperature in which most product was produced in the time interval we earlier determined. We analysed our results and came to the conclusion that the optimal temperature for AP's activity was 40oC. The purpose of the next experiment, experiment 3, was to characterise the enzyme by applying Michaelis-Menten kinetics. We measured the velocity of product formation (Vo) over a substrate interval ranging from 0.05mM - 0.5mM, and determined Michaelis-Menten constants Vmax, Km and Kcat for the AP enzyme.


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