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Effect of an Increasing Substrate Concentration on Enzyme Activity Rate

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Hui Tzu(Erin) Wang


Effect of an Increasing Substrate Concentration on Enzyme Activity Rate


The reaction rate of an enzyme can be affected by many factors, and the purpose of this experiment was to find out how an increasing substrate concentration influences the rate of an enzyme activity; we obtained data from recording the absorbance of the samples which contain the same amount of potato juice (enzyme oxidase) and different amount of catechol (substrate) while holding pH and temperature constant. Our findings illustrate that the rate of enzyme activity is only influenced by substrate concentration at low level of substrate concentration, and as substrate concentration increases, the rate becomes independent of the substrate concentration and proceeds at a constant state.


The fact that enzymes function as biological catalysts has a significant value in millions of living organisms including human beings. As catalysts, enzymes, which are mostly proteins, speed up various reactions by lowering the activation energy they required. The mechanism behind enzyme catalysis involves the binding of substrate to the active site of the enzyme, which proceeds to form an enzyme-substrate complex by process called ÐŽ§induced fitЎЁ and destabilizes the chemical bonds of the substrate, therefore reduces the energy needed for the reactants to reach the transition state (intermediate). The rate of enzyme reaction can be affected by many factors such as temperature, pH, and substrate concentration; every enzyme has its optimum range of temperature and pH, outside that range the enzyme is rendered inactive and is said to be totally inhibited(Logan, 1996). But how is the catalytic rate of an enzyme related to the substrate concentration?

With the purpose to understand the effect of substrate concentration on the rate of enzyme activity, in this experiment we obtained samples which contain the same amount of enzyme and each sample was added with different amount of substrate, than we made observation of the reaction rate in timed intervals. The data obtained would be able to tell us how enzyme reacts to different substrate concentration during a period of time.


Prior to the experiment , the following materials were prepared: 1)the extract of Solanum tuberosum(potato juice) which contains the enzyme catechol oxidase is prepared by blending potato and distilled water at high speed filtering the resulting liquid through several layers of cheese cloth. 2) A pH 7 phosphate buffer, 3) substrate catechol.

First we obtained and labeled five test tubes with numbers 1 to 4 and a B (which indicates blank), and add phosphate buffer and catechol as indicated in Table 1 below. We add 1.5 mL of Potato juice to tube B, cover the tube and invert it several times to mix the contents well. The tube B will be used as blank to adjust the spectrophotometer to absorbance every time before placing the tubes into the spectrophotometer. Then we add 1.5mL catechol into tube 1 and mixed the contents; the absorbance is immediately measured by the spectrophotometer (at 420nm) and recorded and we made a measurement of the absorbance and recorded the numbers every 2 minutes for 10 minutes. For test tubes 2 to 4 we repeated the same steps we performed on test tube 1.

Table1. Protocol for preparing test tubes.

Tube/Trial pH Phosphate buffer Catechol

Blank 6.9mL None

1 6.8mL 0.1mL

2 6.5mL 0.4mL

3 5.7mL 1.2mL

4 5.3mL 1.6mL


The obtained results are showed by Table 1 and we were able to generate Figure 1from the results to show a graphical demonstration of the data collected.



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