How Varying Temperature Affects The Rate Of An Enzyme

Effect of Varying Temperatures: The enzyme catalyzed reaction rate during varying incubation temperatures are plotted on Figure. 6. As the temperature increases the rate increases, but as the temperature reaches 49oC it begins to drop. When the plot of the logarithm of the rate is used against the inverse of the temperature kelvin’s the Arrhenius equation is used to calculate the activation energy. The range in orange is between 16.5 - 37oC and the activation energy is calculated to be 9332kcal/mol.

Discussion: The dilution made for all the assays was 8x which falls between an absorbance of 0.5-1.0 when measured at 405nm. On Figure. 1, it shows the variation of rate as a function of the weight of alkaline phosphatase. In the graph, the rate
3), we see an increase of the rate as the pH reaches pH10. Once pH10 is reached, the reaction rate begins to decrease as the pH increases. This tells us that the optimal pH for the enzyme is pH 10 which gives us the highest µmoles/min/mg of protein. When the phosphatase assays are made with varying substrate concentrations, both a reaction rate vs. substrate concentration and a Lineweaver-Burk plot was made. However, the Vmax and Km couldn’t be determined in the first plot because it is difficult to extrapolate data from a curve, and it is hard to tell where the velocity begins to limit itself. However, when a linear Lineweaver-Burk plot is made, it is easy to extend and extrapolate data from a linear plot which would give us an equation to calculate both Vmax and Km. Therefore, Lineweaver-Burk plot would give more precise Vmax and Km
In addition, the Km is lower in the assays without phosphate because without an inhibitor, the enzyme can be half saturated at a lower concentration as oppose to having inorganic phosphate. This concludes that inorganic phosphate is a competitive inhibitor. If phosphate was non-competitive the Vmax would be decreased and the Km would stay the same and if it was an uncompetitive inhibitor both Vmax and Km would decrease the same amount. Since the inorganic phosphate binds by competitive inhibition, it means that it can bind reversibly to the active site of alkaline phosphatase and compete with the substrate for that site. With the Km values of both with and without inorganic phosphate, the Ki is found to be 0.45mM. This value is smaller than the Km value without inorganic phosphate at 0.38mM. Since the Km is smaller than the Ki, the inorganic phosphate is not very efficient in inhibition because it is half saturated at a higher concentration than the normal substrate. This tells us that the inorganic phosphate more readily dissociates from the enzyme than the normal substrate. For inorganic phosphate to be an effective competitive inhibitor, the Ki must be much lower than the Km. In future experiments a stronger competitive inhibitor with a lower Km can be used to study the effects of a competitive inhibitor on alkaline