Enzyme Kinetics

Fully guided procedure

Standard preparation
Substrate concentration effect
Temperature effect

Standard reference
1. Prepare starch solutions from the stock solution
(1.0 mg/ml) into dilutions of 0.01, 0.025, 0.05,
0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the
starch stock solution.
2. Iodine solution is prepared by adding 5 g
potassium iodide to 100 ml water. The
dissolved potassium iodide is added with 1 g of
iodine and is allowed to dissolve.
3. Prepare a standard curve of Absorbance (@
590 nm) vs. Concentration of a starch/iodine
mixture. Use the following table as guide.

Substrate concentration
effect

Data analysis
1. Calculate starch concentration for each sample after hydrolysis (S F)
through use of the standard curve. The initial starch concentration (S 0) is
already known. [S] = (So) (SF)
2. The velocity (rate of digestion) of the reaction for each sample can be
calculated as:
3. V = S/ t = (S0 - SF) / 10 minutes
4. Prepare a table showing rate of hydrolysis (V) at different the starch
concentrations.
5. Plot a Michaelis-Menten graph.
6. Prepare a graph of 1/starch concentration (x-axis) versus 1/rate of
digestion (y-axis). This type of reciprocal graph displaying enzyme
kinetics is a Lineweaver-Burke plot.
7. State the value of Vmax and Michaelis constant Km from your graph.
8. The y-intercept of the Line-weaver Burke plot is the reciprocal of the
maximum velocity of the reaction (V max). The x-intercept is the negative
reciprocal of the Michaelis constant. (K m).

Table to guide
S0

Sf

V=S/t

1/S0

1/V

Temperature effect

Data analysis
1. Plot the Lineweaver-Burke line for
the result of 20, 28, 35 and 40C.
2. Compare all three plots.
3. What are the values of Vmax and Km
for all plots?