The effect of substrate concentration on enzyme activity

Determination of Rate of Enzyme Activity of Catalase
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Determination of Rate of Enzyme Activity of Catalase
Background
Enzymes are organic catalysts that are used to increases the rate of a biochemical reaction. They are mainly made of proteins. However, ribonucleic or deoxyribonucleic acids may also act as enzymes under specific circumstances (Kraut, Carroll, & Herschlag, 2003). Enzymes act by lowering the activation energy of the substrates to form products. Activation energy is defined as the minimum energy required for substrates to combine, and get converted to its respective products. Enzymes lower this activation energy and speeds up a biochemical reaction (Stroppolo et al., 2001). Enzyme activity is defined as the potency of the enzyme, in converting substrates into their respective products, as a function of time (Danson &Eisenthal, 2002).
Therefore, less the time required and more the amounts of substrate converted/or products formed indicates that the enzyme activity for a particular enzyme is high. The enzyme acts through catalytic sites that are known as active sites. These sites can undergo conformational changes, which helps the substrates to combine easily, to form the respective products (Hammes, 2002). Enzyme activity depends upon various factors. These include concentration of substrates, temperature and pH conditions, under which the reaction takes place (Danson &Eisenthal, 2002).

The reaction of an enzyme to its substrate is governed by Michaelis-Menten’s hyperbolic kinetics (Beal, 1983). Hyperbolic kinetics indicates that rate of enzyme activity increases with increased concentration of the substrates. However, when the active sites are saturated with substrates rate of the reaction do not increase further and is maintained at the steady state (Beal, 1983). Enzyme activity also changes when temperature conditions and pH of the reaction is altered. Each enzyme shows an optimum activity under a certain range of temperature or pH change. However, beyond that range the enzyme activity falls and decreases. This is because increased temperature may denature the active site of the enzyme. The loss of three- dimensional conformation of the active site would cause functional loss of an enzyme activity. Similarly, a change in pH may lead to a loss of charged profile of the active site, causing functional loss of enzyme activity. The present experimentation was performed, to study the enzyme activity of catalase under varying substrate concentrations, and also under different temperature and pH conditions.
Principle of the Enzymatic Reaction (Assay)
The present experimentation was performed to study the enzyme activity of catalase. Catalase is an enzyme that is found in various plants and animals. These enzymes scavenge reactive oxygen species like hydrogen peroxide and detoxify the body of an organism. Reactive oxygen species are detrimental to the cells of an organism. They are associated with damage to the cell membrane and DNA. Damage to these organelles is associated with various pathological outcomes. Catalase reacts with Hydrogen peroxide and converts it into water and oxygen (Chelikani, Fita,& Loewen, 2004).
The assay that was implemented in this study was to assess the rate of enzyme activity of catalase. In the experiment, a filter paper was soaked in an aqueous extract of catalase (potato extract). Hydrogen peroxide was used as the substrate. When the enzyme breaks down hydrogen peroxide, oxygen is formed and pushes the filter paper at some distance. The distance moved by the filter paper as a function of time, represents the rate of enzyme activity (Chemistry Lab Manual). The Rate of enzyme activity is expressed as below:
Rate of enzyme activity = Distance (depth of hydrogen peroxide in mm)/Time (in seconds)
Methodology
Potato extract was prepared as a source of catalase. 30 gram of potatoes were blended in 60 ml ice water and filtered. Different concentrations of hydrogen peroxide were taken as the substrates. The concentrations of hydrogen peroxide were (0.1%, 0.3%, 0.5%, 1%, 2%, 3%). Filter papers (Whatman No.1) with 2.5 com diameter were taken for soaking the catalase extract. The hydrogen peroxide concentrations were tabulated in Table 1. Three beakers were set up with 0% hydrogen peroxide (control substrate, which was water). As mentioned forceps were used to draw the paper circles and excess potato juice was wiped from the filter paper (Chemistry Lab Manual).
The filter paper was then placed with forceps in the beaker containing hydrogen peroxide (in different concentrations). A stop watch was used to measure the time, which was taken for the filter paper to reach the surface. The timing was noted as soon as the paper was introduced in the beaker (Chemistry Lab Manual). The recordings are exhibited in Table 1. The same experiment was repeated for different concentrations of hydrogen peroxide and also thrice for each concentration. The average time for each concentration was plotted. The enzyme activity was measured from the depth of hydrogen peroxide (mm) as a function of time. Enzyme activity was calculated for each concentration and was plotted in a graph (Fig 1).
Results
Conc. of H2O2 Depth of H2O2 in mm Time in seconds
a Time in seconds
b Time in seconds
c Mean Time in seconds Enzyme activity mm/sec
0% 0.1% 30 mm 144 s 123 s 162 s 143 s 0.209
0.3% 30 mm 66 s 60 s 83 s 69.66 s 0.428
0.5% 30 mm 55 s 47 s 65 s 55.66 s 0.535
1.0% 30 mm 40 s 35 s 48 s 41 s 0.731
2.0% 30 mm 29 s 16 s 23 s 22.66 s 1.304
3.0% 30 mm 15 s 17 s 20 s 17.33 s 1.764
Table 1: Represents the estimation and Readings of enzyme activity of catalase under different concentrations of hydrogen peroxide.

Fig1: Represents the catalase activity concerning time.
Discussion and Conclusion
From the results, it was evident that with the increase in substrate concentration (increasing hydrogen peroxide concentration) the rate of enzyme activity increased in a non-linear manner (Duggleby, 1995). This was because the active sites of catalase were increasingly occupied by increased concentrations of the substrate. Therefore, as per Michaelis-Menten’s hyperbolic kinetics the rate of enzyme activity was directly related to substrate concentration, which was evidenced in the present assay also. However, unlike the hyperbolic kinetics, no steady state was observed with the substrate concentrations, which were considered for the assay. This implied that perhaps the active sites of catalase was not fully occupied or saturated with the concentrations of hydrogen peroxide considered in the study. Nevertheless, the assay provided the evidence; that catalase is an effective scavenger of free radical species. Increased catalase concentration within plants or animal tissues may protect the cells from oxidative stress. There were some concentrations, where the enzymatic activity drastically changed. This might be possible if hydrogen peroxide exhibited some form of allosteric effect at certain concentrations. This means that binding of a certain concentration of hydrogen peroxide to one of the active sites of catalase stimulated binding of more concentrations of hydrogen peroxide to other active sites of catalase (Ricard & Cornish Bowden, 1987). Such bindings may have occurred very rapidly, which was not possible at lower concentrations of hydrogen peroxide.
Reference
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