Enzymes - investigate how the substrate concentration (H2O2) affects the activity of catalase on hydrogen peroxide.

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Sheyam Patel

Enzymes

Aim

To investigate how the substrate concentration (H2O2) affects the activity of catalase on hydrogen peroxide.

Introduction

Enzymes are protein molecules, which can be defined as biological catalysts. A catalyst is a molecule that speeds up a chemical reaction, but remains unchanged at the end of the reaction. An enzyme catalyses virtually every metabolic reaction, which takes place within a living organism.

Enzymes have a globular protein structure. The enzyme molecules are coiled into a precise three-dimensional shape, with hydrophillic R groups (side chains) on the outside of the molecule ensuring that they are soluble. Enzyme molecules also have an active site, which is usually a cleft of depression, to which another molecule or molecules can bind. This molecule is the substrate of the enzyme. The shape of the active site allows the substrate to fit perfectly, and to be held in place by temporary bonds, which form between the substrate and some of the R groups of the enzyme’s amino acids. The combined structure is termed the enzyme-substrate complex.

Each type of enzyme will usually act on only one type of substrate molecule. This is because the shape of the active site will only allow one shape of molecule to fit. The enzyme is said to be specific for this substrate.

 

The enzyme may catalyse one of two types of reactions. One in which the substrate molecule is split into two or more molecules; the other in which it may join two molecules together, as when making a dipeptide. Interaction between the R groups of the enzyme and the atoms of the substrate can break, or encourage formation of, bonds in the substrate molecule, forming one, two or more products.

The product or products leave the active site when the reaction is complete. The enzyme is unchanged by this process, so it is now available to receive another substrate molecule. The rate at which substrate molecules can bind to the enzyme’s active site, be formed into products and leave can be very rapid. This theory of how enzymes work

Is known as the lock and key theory. A substrate

Lock and key theory (Biology 1, 2002)

molecule is a ‘key’, and binds to the active site of an enzyme, a ‘lock’.

The induced fit theory is another theory of how enzymes work. The active site of an enzyme is a cavity of a particular shape, and initially the active site is not the correct shape in which to fit the substrate. As the substrate approaches the active site changes and this results in it being a perfect fit. After the reaction has taken place, and the products have gone, the active site returns to its normal shape.

Induced fit theory (Biology, John Parker, 2000)

As catalysts, enzymes increase the rate at which chemical reactions occur. Most of the reactions that occur in living cells would occur so slowly without enzymes that they would virtually not happen at all. In many reactions, the substrate will not be converted to a product unless it is temporarily given some extra energy. This energy is called activation energy. Enzymes speed up a chemical reaction by reducing the activation energy. The rate of a reaction is at its optimum due to a number of factors. The course of a reaction is affected by a number of factors, which are enzyme concentration, substrate concentration, temperature and pH.

Temperature

Temperature is a factor that affects enzyme activity. At low temperatures, the reaction takes place only very slowly. This is because molecules are moving relatively slowly. Substrate molecules will not often collude with the active site, and so binding between substrate and enzyme is a rare event. As temperature rises, the enzyme and substrate molecules move faster. Collisions happen more frequently, so that substrate molecules enter the active site more often. Moreover, when they do collide, they do so with more energy. This makes it easier for bonds to be broken so that the reaction can occur.

As temperature continues to increase, the speed of movement of the substrate and enzyme molecules also continues to increase. However, above a certain temperature the structure of the enzyme molecule vibrates so energetically that some of the bonds holding the enzyme molecule in its precise shape begin to break. This is especially true of hydrogen bonds. The enzyme molecule begins to lose its shape and activity and is now denatured. This is often irreversible.

At first, the substrate molecule fits less well into the active site of the enzyme, so the rate of reaction begins to slow down. Eventually the substrate no longer fits at all, or can no longer be held in the correct position for the reaction to occur. The temperature at which an enzyme catalyses a reaction at the maximum rate is called the optimum temperature. Most human enzymes have an optimum temperature of around 40°C. by keeping our body temperatures at about 37°C, we ensure that enzyme-catalysed reactions occur a close to their maximum rate. It would be dangerous to maintain a body temperature of 40°C, as even a slight rise above this would begin to denature enzymes.

pH

Enzymes are also affected by pH. Most enzymes work fastest at a pH of somewhere around 7, that is in fairly neutral conditions. Some, however, such as the protease pepsin which is found in the acidic conditions of the stomach, have a different optimum pH.

pH is a measure of the concentration of hydrogen ions in a solution. The lower the ph, the higher the hydrogen ion concentration. Hydrogen ions can interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their three-dimensional arrangement. A ph that is very different from the optimum pH can cause denaturation of an enzyme.

Enzyme concentration

When considering the rate of an enzyme catalysed reaction the proportion of enzyme to substrate molecules should be considered. Every molecule fits into an active site, and then the reaction takes place. If there are more substrate molecules than enzyme molecules, then the number of active sites available is a limiting factor. The optimum rate of reaction is achieved when all of the active sites are in use. At this stage, if more substrate is added, there is no increase in the rate of product formation. When there are less substrate molecules than enzymes the reaction will take place very quickly, as long as the conditions are appropriate.

Substrate concentration

As substrate concentration increases, the initial rate of reaction also increases. Again, this is only what we would expect: the more substrate molecules there are around, the more often an enzyme’s active site can bind with one. However, if we go on increasing substrate concentration, keeping the enzyme concentration constant, there comes a point where every enzyme active site is working continuously. If more substrate is added the enzyme simply cannot work faster: substrate molecules are effectively ‘queuing up’ for an active site to become vacant. The enzyme is working at its maximum possible rate, known as Vmax.

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Catalase is an enzyme that catalyses the reaction by which hydrogen peroxide is decomposed to water and oxygen. 2H2O2  2H2O + O2

Found extensively in mammalian tissues, liver, muscle (beef), apple and potato. catalase prevents the accumulation of and protects the body tissues from damage by peroxide, which is continuously produced by numerous metabolic reactions. In humans hydrogen peroxide is catalysed by catalase in the liver. Hydrogen peroxide needs to be broken down because it is poisonous.        

Preliminary experiment

I carried out a preliminary experiment in order to familiarise myself with the procedures of this experiment. To find ...

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*** A well constructed report that contains usable data. There are some technical issues with regard to the recording of data and details of the methodology that could be improved. To improve: Research and rationale The background research could be extended to include sources that are beyond those of the standard A level texts. The sources quoted should be clearly referenced within the text to illustrate where they have been used. It should be made clear to the reader how the hypothesis and methods used have been developed. Planning Whilst the use of a pilot experiment is always advisable, it needs to be explained to the reader why this was carried out varying temperature and not substrate concentration, as was the intended aim of the experiment. The pilot experiment should be used to inform the planning of the investigation. The candidate needs to take care that key technical terms are used correctly. e.g dependent and independent variables. The preparation or source of the catalase was not explained in any way and this has an impact on the interpretation of the experiment. The control of the key variables could be improved by using a thermostatically controlled water bath and a buffer. The risk assessment would not be considered adequate under current guidelines. Implementing The candidate seems too have used the apparatus competently to collect usable data but results were not recorded in suitable tables with clear headings. Some of the headings were incorrect. Units should be clearly indicated in the headings only and should follow the Institute of Biology guidelines. Analysis and Evaluation. The summary tables were presented in a format that did not present the results as clearly as possible. The inclusion of a table showing the initial rates of reaction for each substrate concentration would have been helpful. The graph was not included. The limitations could be discussed in more depth as they are concerned mainly with basic errors. The candidate is reasonably aware of the lack of precision in the experiment and has made some sensible suggestions for improvement. Some proposals for extending the scope of the work would be helpful.