Enzyme Catalyzed Reaction Kinetics Table 1 shows the results of an investigation of the effect of substrate concentration on the rate of an enzyme-catalyzed reaction.
A series of identical reaction tubes were prepared using a buffer solution at the optimal pH for the enzyme. A standard amount of enzyme was added to each tube so they were all at the same concentration and the tubes equilibrated in a water bath at the optimal temperature for the enzyme, so the concentration of the substrate was the only variable.
The reaction was initiated by addition of an equal volume of substrate at the appropriate concentration (staggered at 30 second intervals). The reaction was terminated after 10 minutes by addition (at 30 second intervals) of an inhibitor. The amount of reaction product was determined spectrophotometrically.
Table 1: Kinetic data showing variation in the effects of substrate concentration on the rate of an enzyme catalyzed reaction
Substrate concentration [S]
(mmol.l-1)
0
2
4
8
12
16
20
24
Reaction rate
(v)
(mmol.min-1 x 10-2)
0
6.5
9.6
12.4
14.1
14.7
14.9
14.9
Enzyme Catalyzed Reaction Kinetics Instructions
1Plot a graph to show the effect of substrate concentration on the rate (velocity) of the reaction.
2From the graph, determine values for the maximal velocity (Vmax) and the half-maximal velocity (½Vmax)
3 Use the latter value to determine the substrate concentration which causes ½Vmax. This value is the ‘Km value’ or the ‘Michaelis constant’. Using on the results comment on the measure of the affinity of the enzyme for the substrate i.e. the amount of enzyme that is required to produce half the maximal velocity of the reaction.
4 Plot a graph of the 1/[S] and 1/v and from the graph determine Vmax and Km (this is Lineweaver-Burke reciprocal graph). Using on the results comment on the measure of the affinity of the enzyme for the substrate i.e. the amount of enzyme that is required to produce half the maximal velocity of the reaction.
5 Using researched literature and your results discuss the difference in the results of Vmax and Km that you have found in the two methods.
6 Decide if any of the two methods are accurate/which is best accurate method for the calculation of Vmax and Km.
7 Identify the main variables (independent, dependent and control variables) in this experiment.
