FACTORS AFFECTNG THE RATE OF ENZYMATIC REACTIONS
M. B. Mncwango
University of KwaZulu-Natal, School of Life Sciences, Westville Campus, Durban, 4001
Enzymes are biological catalysts in almost all metabolic reactions, of which they primarily have a function to lower the activation energy in a metabolic reaction. Enzymes act upon substrates to convert into a different molecule known as products (Murphy et al. 2014). The study of enzymes is called enzymology. Enzymes have three dimensional structures. Enzyme activity can be affected by other molecules i.e. inhibitors (molecules that decrease enzyme activity e.g. therapeutic drugs, poisons etc.), activators (molecules that increase enzyme activity). The activity of an enzyme will decrease when it is not within range of the optimum temperature or pH. In this state enzymes are said to have denatured (Dunaway-Mariano.2008).
This whole experiment consists or two parts (experimental processes) namely: Experiment 1 and Experiment 2. In Experiment 1, we investigate the effect of pH on the rate of enzymatic reactions. In Experiment 2, the effect of substrate concentrations on the rate of enzymatic reaction is investigated. Ofcourse there are other factors that affect the enzymatic rate of reaction like enzyme concentration, temperature, activators and inhibitors but in this experiment only two factors are investigated i.e. pH and substrate concentration.
Methods and material
A catalase extracted from young spinach leaves was given by homogenizing 20g of the leaf tissue in 100ml buffer of pH 7.2. The homogenate was centrifugated at 2,000 × g for 5 minutes. A resulting supernatant liquid was used for the experiment. A macro-manometric apparatus was assembled. The enzyme extract and buffer were mixed each time an experiment was ran. The reaction vessel had to be mounted and assembled on a retort stand with a clamp and immersed the reaction vessel into a water bath with a constant temperature of 30?. The reaction vessel was connected to the macro-manometric apparatus with the rubber and glass tubing. The macro-manometric apparatus was zeroed by using the pinch clamp and was closed off. Approximately 3 minutes was allowed for thermal equilibration between the contents of the reaction vessel and the water bath. Hydrogen peroxide (H_2 O_2), the substrate, was instantaneously added at the 3 minutes and the vessel was closed tightly.
The reaction vessel was agitated continuously and gently. The volume of oxygen collected in the manometer during the 5 minutes duration was carefully noted. The effect of pH on the rate of enzymatic reaction was investigated by preparing buffer solutions of pH 4.0; 5.0; 5.4; 6.0; 6.6; 7.2; 7.8; 8.4; 9.0; 10.0. 20 ml of each buffer solution was placed in different test tubes and 2.0ml of the extract was added to each test tube. The contents of one test tube was transferred to the reaction vessel. Thereafter 10ml of 3% H_2 O_2 and the reaction rate was determined. The reaction vessel was washed and rinsed. The same was done for all the other pH in the different test tubes. The results (the calculated reaction rates for each test tube) were listed and used for the graph of reaction rate versus pH and the optimum pH was calculated for the catalase activity.
Seven different H_2 O_2 solutions were prepared in test tubes and placed on ice. The test tubes were clearly labelled beforehand. The reaction was allowed to proceed just like before in Experiment 1 above. The reaction rates were calculated and used for the plotting of the graph of reaction rate versus substrate concentration
Test tube number
3% H_2 O_2 (ml) Buffer A (ml) H_2 O_2 Concentration
1 0 10 0
2 0.5 9.5 0.15
3 1 9 0.3
4 2 8 0.6
5 4 6 1.2
6 8 2 2.4
7 10 0 3
Table 1: Shows the protocol for preparing the different H_2 O_2 solutions to determine the effect of substrate concentration on enzyme activity.
The final H_2 O_2 concentration was calculated using the following method:
C1 is a constant given by 3
V1 = 3% H2O2 (ml)
C2 is the unknown H2O2
Figure 1: The graph shows the reaction rate versus the pH (Experiment 1).
The graph in Figure 1 shows a curvilinear correlation, as it first shows a positive linear correlation and thereafter shows a negative correlation. As the graph increases it has a steep gradient and as it decreases its gradient tends to be gentler and gentler. The graph has a trendline given by the equation y=-0,7911x^2+6.0861x-4,77
Figure 2: The graph shows the reaction rate versus substrate concentration that was investigated in Experiment 2.
The graph has a positive correlation. It has a gradient that can be described as gentle but not too gentle. A trendline with the equation: y=1,648x-1,52 can be drawn for the graph illustrated in Figure 2.
When observing the oxygen collected in the manometer during the succeeding 5 minutes, or the time required to collect 40ml of oxygen, if less than 5 minutes. A desirable reaction rate is approximately 10ml per minute. If the observed activity in the trial run exceeds this rate, the enzyme extract should be diluted with deionized water (distilled water) before proceeding with the experiment. This step is taken to ensure that the 5 minutes in the experiment is exhausted. One may ask may should distilled water should be used specifically? Why isn’t normal tap water used to dilute the extract. Well the reason for this is because water from the tap or any other water for this instance have ions whilst distilled water doesn’t have any, hence the name deionized water. The ions contained within the other kinds of water will alter with the reaction that will be occurring in the reaction vessel. To ensure that the results are reliable and valid every time a 20ml of buffer solution measured was pipetted using a different measuring pipette to avoid contamination between the buffer solutions. Figure 1 suggests that the optimum pH for enzymatic reaction is at the pH of 7.2. The hypothesis is therefore not rejected