The Effect of Substrate Concentration on the Rate of Reaction with Amylase

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The Effect of Substrate Concentration on the Rate of Reaction with Amylase

Hypothesis:

Null: - There will be no change in the rate of reaction, even with a change in substrate concentration.

Alternative: - The rate of the enzyme-catalysed reaction will occur at a faster rate with an introduction of a higher concentration of substrate, and the rate of reaction will only be bounded by the availability of active sites.

Introduction:

The aim of this experiment is to show that the substrate concentration affects the rate of reaction, using the enzyme amylase and different concentrations of starch substrate.

Similar to cellulose, starch molecules are glucose polymers linked together by the alpha-1, 4 and alpha-1, 6 glycosidic bonds, as opposed to the beta-1, 4 glycosidic bonds for cellulose. Starch is composed of two polymers, the linear amylose and the highly branched amylopectin.

 In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylase, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose unit.

Alpha-amylase will be used in the experiment. This hydrolyses alpha 1.4 glycosidic bonds within starch to form smaller sugars. It is important to know we are using alpha-amylase and not beta- amylase. The latter will only produce disaccharides, as it is an alternate enzyme. Human saliva and pancreatic secretion contain a large amount of alpha-amylase for starch digestion. Amylase is a tertiary-structured protein, which means it has a globular shape therefore making it a globular protein.

Proteins are made up of amino acids, which are monomers. Condensation reactions between amino acids result in peptide bonds. When more amino acids join together via peptide bonds it creates a chain of monomers, or a polymer. This is called a polypeptide chain. The polypeptide chain is then folded, branched and linked at different areas, which gives it a globular shape, becoming a protein.

These enzymes are known as organic catalysts. They speed up metabolic processes, acting as a cataboliser, i.e. breaking the larger molecules, namely starch, down into the smaller molecules, small enough so that the body can absorb them. Enzymes have specialised active sites, which allow them to attach to specific parts of molecules, and detach it from the rest of the molecule, breaking the glycosidic bonds in the process. As the enzymes are organic catalysts this means it does not get ‘used up’, and can therefore be used again.

When an enzyme joins to the substrate, it is known as the enzyme-substrate complex. There are two main theories surrounding the enzyme-substrate complex. The ‘lock and key’ theory and the ‘induced fit’ theory:

An enzyme can only catalyse one reaction at a time. So therefore catabolism of a large amount of starch, will take time. The recognised value for the number of substrate molecules an enzyme can react with in one second is known as the turnover number.

This investigation will show the relationship between the substrate concentration and the rate of enzyme action upon the substrate. Results will be recorded in a qualitative fashion, noting the colour change of the enzyme substrate mixture, when added to the iodine.

Factors:

There are two variables, which need to be taken into consideration. The independent variable and the dependent variable. The independent variable is manipulated by the experimenter; in this experiment it is the substrate concentration. The dependent variable, on the other hand, should theoretically change in accordance to the value of the independent variable. In this experiment the dependent variable is the change in colour of the iodine when the enzyme-substrate mixture is added.

Temperature of the enzymes:

An enzyme will react with its substrate at a different rate if the surrounding temperature is changed. All enzymes have an optimum temperature, at which the rate of reaction is fastest. At this optimum temperature, approx. 60 degrees centigrade for alpha amylase, the enzyme will have its maximum kinetic energy possible, without having denatured. This means that the enzyme will move around more freely and successful collisions are more likely to occur. In turn this means that the rate of reaction will increase, i.e. the substrate will be broken down into smaller units more quickly.

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As mentioned above, an enzyme can denature if the surrounding temperature is too high. Surplus heat energy disrupts bonds in the enzyme. This means the structure of the enzyme will be changed, including the shape of the active site. If the active site is changed then the enzyme can no longer interact with the substrate. This is the reason for consideration, and is also the reason that the water bath, regulated at 60 degrees centigrade will be used for these experiments. Both the substrate and the enzyme will be placed in the water bath before the experiment, in separate test ...

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