The major experiments that are conducted in the determination of enzyme kinetics are those that vary the amount of concentration in order to measure the enzyme velocity . The aim of such experiments is to determine the Km or the amount of the substrate that gives half the enzyme velocity, as well as the Vmax, which is the maximum velocity attained by the enzyme . In addition, the enzyme kinetics may be presented graphically using the Eadie–Hofstee diagram. The diagram made by plotting the rate of reaction as a function of the ratio between rate and concentration of the substrate as shown in the equation below.
Plotting v against v/[S] results in the determination of Vmax by calculating the y-intercept and Km, which is the negative slope .
The experiment aimed at determining the Michaelis constant, as well as the maximum velocity of a reaction.
The spectrophotometer was switched on and ensured that it was set in absorbance mode at 420nm. The volumes of the substrate stock and buffer needed were calculated using the formula (C1V1=C2V2) to provide solutions of a final volume of 8 ml containing 2000, 1500, 1000, 500, and 250 µM BAPNA. The values were entered in Table 1 below and dilutions performed.
Aliquot of 2 ml from each stock tube was taken and added into two clean tubes, one for each temperature needed to perform the reaction, 370C and 650C. The tubes were labeled with BAPA concentration and temperature. One milliliter of enzyme was added to each tube at 30s intervals, and the content mixed immediately after adding the enzyme by swirling. The tubes were then incubated for 45 minutes at 370C and 650C. After 45 minutes, 0.5 ml of acetic acid was added to each tube at an interval of 30s to stop the reaction and the content mixed by swirling. Ensuring that the spectrophotometer was set at 420nm, a cuvette containing buffer was placed in the spectrophotometer, and the absorbance measured. The sample was used to zero the spectrophotometer. In the same way, the absorbance for each tube was measured and recorded.
The calculated values for the concentration of BAPNA and the absorption at 370C and 650C were recorded in table 2 below.
The Michaelis-Mentos plot had a significant accuracy in the determination of Km and Vmax although other methods such as nonlinear regressions are more accurate. The Eadie–Hofstee also gave Km and Vmax values that are similarity accurate. The value of Km shows the amount of the substrate that gives half the enzyme velocity and Vmax gives the maximum velocity attained by the enzyme . The effect on the rate at which the reactions occurred by temperature was evident when the two reactions were compared. The reaction that at 650C had a markedly reduced rate of reaction compared to the reaction at 370C possibly due to enzyme denaturation by high temperatures. The rate of reaction in both reactions, however, increased as the level of substrate concentration increased. The increased rate at increasing substrate concentration may have been as a result of more substrates interacting with the enzyme.
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