Investigate the effect of pH on the rate of enzyme-controlled reactions.

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Christopher Magee                                                                 Wednesday 15th January 2003

PLAN: Investigate the effect of pH on the rate of enzyme-controlled reactions 

Aim: The specific enzyme that will be examined and utilized within this investigation is ‘catalase’ (present within a quantity of potato extract). In order to determine the enzyme’s optimum pH a series of experiments must be carried out where the pH concentrations (buffer solutions) are changed, while the substrate (hydrogen peroxide) and enzyme concentrations remain constant.

Hypothesis, Prediction and Scientific Background: 

Catalase: an enzyme, found in peroxisomes, which catalyses the decomposition of hydrogen peroxide, which results from oxidation reactions in the cell; to yield water and oxygen. High concentrations of catalase are found in the liver. Furthermore catalase is the fastest known enzyme and an important part of the body’s antioxidant defences.

Therefore catalase is a protein that, in small amounts, speeds up the rate of a biological reaction without itself being used up in the reaction (i.e. it acts as a catalyst). The enzyme acts by binding with the substance involved in the reaction (the substrate – hydrogen peroxide) and converting it into another substance (the product of the reaction – water and oxygen). As the enzyme is relatively specific in the type of reaction it catalyses; hence there are many different enzymes for the various biochemical reactions.  The enzyme requires certain pH conditions for optimum activity; where a maximum and efficient amount of products are formed.

Therefore catalase is an enzyme that brings about (catalyses) the reaction by which hydrogen peroxide is decomposed to water and oxygen. It is found extensively in mammalian tissues, catalase prevents the accumulation of and protects the body tissues from damage by peroxide, which is continuously produced by numerous metabolic reactions.

pH: a scale, which measures the concentration of hydrogen ions in a solution. Solutions with pH values below seven contain high concentrations of hydrogen ions and are acidic. Solutions with values above seven have low concentrations of hydrogen ions and are alkaline. The pH of the surrounding medium can have a marked effect on the bonding responsible for the secondary and tertiary structure of the globular protein molecules and hence on enzyme-controlled reactions.

The enzyme catalase carries out the following reaction:

Catalase uses the iron atom to help it break the bonds in the two molecules of hydrogen peroxide and converts them to two water molecules plus a molecule of oxygen gas. Hydrogen peroxide is a toxic molecule that is created in our bodies during normal metabolic events, but as catalase is present in the peroxisomes of the majority of human cells, it serves to protect the cell from any toxic effects by catalysing the decomposition of H2O2 without the production of oxygen free radicals.

The pH of a solution can have several effects of the structure and activity of enzymes. For example, pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive.

The formation of the enzyme-substrate complex depends on a precise match of shape and charge. If there is a change in pH, which will cause a change in the amount of free H+ or OH- ions, it will disrupt these charges. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis.

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 At the optimum pH, the reaction occurs very fast. The maximum catalytic rate for one catalase molecule is 6 million molecules of hydrogen peroxide converted to water and oxygen per minute. The reaction product is 6 million molecules of water and 3 million molecules of oxygen. (Because the oxygen molecule consists of two oxygen atoms, the number of oxygen molecules made in the reaction is half the number of water molecules.)

However as enzyme activity is affected by pH, I predict the optimum pH of catalase is approximately pH 7.0 (neutral) this is because if the buffer solution is too ...

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