Chapter 4 Enzymes

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the substrate Products 1 r Enzyme Specificity and the lock and key hypothesis The specificity of an enzyme refers to its ability to catalyse just one reaction or type of reaction. Only one particular substrate molecule will fit into the active site of the enzyme molecule. This is because of the shape of the active site. The shape of the active site is caused by the specific sequence of amino acids. This produces a specific tertiary structure - the three-dimensional shape of the molecule. This is referred to as the lock and key hypothesis. Catalysing the reaction Enzymes can speed up the rate of a reaction at body temperature. They lower the activation energy required for the reaction to occur. The activation energy is the amount of energy required to set off the reaction and break the bonds in the substrate molecule. The induced-fit hypothesis enzyme + substrate Enzymet activity Enzyme denatures as pH decreases below the optimum. At pH 3 enzyme is fully denatured. E+S ESC Figure 5.2 An enzyme-controlled reaction Enzyme activity The induced-fit hypothesis helps to explain how the activation energy may be reduced. The active site of an enzyme molecule does not have a perfectly complementary fit to the shape of the substrate. When the substrate moves into the active site, it interacts with the active site and interferes with the bonds that hold the shape of the active site. As a result, the shape of the active site is altered to give a perfect fit to the shape of the substrate. This changes the shape of the active site, which also affects the bonds in the substrate, making them easier to make or break (and therefore reducing the activation energy). The course of an enzyme-controlled reaction In an enzyme-controlled reaction, substrate is complementary in shape to the enzyme's active site, therefore the substrate enters the enzyme's active site, combining to form the enzyme-substrate complex (ESC). This destabilises and strains the bonds in the substrate, forming the enzyme product complex. The product is finally leaves the active site and the enzyme is then free to take up another substrate molecule. 0 enzyme- substrate complex Optimum pH Enzyme i is Enzyme is A denaturing denaturing 0 12 3 4 5 6 7 8 9 10 11 PH Figure 5.3 The effect of changing pH on enzyme activity 10 Different enzymes are used in different parts of complex processes such as digestion as enzymes are specific to particular substrates as the shapes of the active site and substrate are complimentary, so only certain substrates will be able to form the enzyme substrate complex and have their bonds destabilised via induced fit to form a product with particular enzymes. Increased kinetic energy increases collisions 20 Optimum temperature enzyme- product complex 30 EPC This affects the tertiary structure of the molecule and so alters the shape of the active site. The shape will no longer be complementary to the shape of the substrate molecule, so the enzyme substrate complex will no longer form, meaning that the enzyme is denatured. The higher the percentage of enzymes that are denatured, the slower the rate of reaction will be. 40 Enzyme is denaturing Enzyme denatures as pH increases above the optimum. At pH 11, enzyme is fully denatured. enzyme + product 60 E + P 50 Temperature/°C Effects of conditions on enzymes pH All enzymes have an optimum pH-the pH at which they work best. Therefore, they will not work as quickly at a pH outside their optimum range. This is because the hydrogen ions that cause acidity affect the interactions between R groups in the tertiary structure of the enzyme, so hydrogen and/or ionic bonds may break. Temperature The effects of temperature change on enzyme action vary Enzyme activity Enzyme activity depending on the temperature range considered. Each enzyme has an optimum temperature at which it is most active. This temperature is often 37°C (in mammals), but it may be different in other organisms. At low temperatures (0-45°C), the activity of most enzymes increases as temperature rises. At low temperatures, the molecules have little kinetic energy. They collide infrequently with the substrate molecules and activity is reduced. As temperature rises, the molecules gain more kinetic energy. They collide more frequently with the substrate molecules and are more likely to have sufficient energy to overcome the required activation energy. Therefore, activity increases. At higher temperatures, the increased kinetic energy causes the enzyme to vibrate, causing vibration within the protein molecule, so hydrophilic and hydrophobic interactions, as well as hydrogen bonds and ionic bonds in the tertiary structure break. This changes the 3D structure/conformation of the protein, so the enzyme loses its shape, becoming denatured. The active site no longer fits the shape of the substrate and activity reduces quickly to zero. Enzyme concentration The effect of pH and temperature on rates is usually because at extremes of pH and temperature. Some enzyme molecules are denatured and the concentration of active enzyme molecules is reduced. Increasing number of active sites occupied at any moment Substrate concentration If the substrate concentration is high, there is a greater chance of successful collisions between the enzyme active sites and substrate molecules, forming enzyme substrate complexes and thus resulting in higher product formation. As the substrate concentration increases, so does the rate of reaction. If the number of enzyme molecules is limited, the rate of reaction plateaus once all the enzyme active sites are fully occupied as enzymes are working at maximum rate. Therefore further increases in substrate concentration will have no effect as enzyme concentration will then be the limiting factor. Enzyme concentration If there are more enzyme molecules in a particular volume of reaction medium, there are more active sites available. There is a greater likelihood of collisions between the enzyme and the substrate molecules. More interactions per second mean a higher rate of reaction. As the enzyme concentration increases, so does the rate of reaction. All active sites occupied at any moment Substrate concentration Practical investigations Effects of pH, temperature, enzyme concentration and substrate concentration You should be familiar with how the effects of changes in the following factors on enzyme activity can be investigated experimentally: • pH • temperature • enzyme concentration • substrate concentration It is important to consider the following points. 1 Volume and concentration of enzyme solution. 2 Volume and concentration of substrate solution. 3 Control of temperature. A thermostatic water bath is often the best way. 4 Control of pH. A buffer solution controls pH. 5 How can you make the test more reliable? Repeat the test a number of times and calculate the mean. 6 Testing reliability. Comparing raw data to the mean is a good indication. There should be little variation around the mean this can be shown using range bars. Calculating the standard deviation is even better. 7 Whether the volume of the solution needs to be fixed or not 8 Distribution of the molecules in the solution. Even distribution can be attained by stirring regularly 9 What is the control? This is a test that omits one factor in the experiment to show that it is essential for the reaction to occur. It is usual to omit the enzyme from the reaction mixture to show that no reaction occurs without the enzyme. 10 What level of precision is appropriate in the measurements? 11 How valid is the experiment? Is it actually measuring what you think it should? Have you taken account of all possible conditions that may affect the reaction rate? Enzyme-controlled reactions Coenzymes and cofactors Coenzymes are larger organic substances that take part in the reaction. They usually transfer other reactants between enzymes. Examples of coenzymes include coenzyme A, which takes part in aerobic respiration, and NAD, which is involved in transporting hydrogen atoms to the inner mitochondrial membrane. Coenzyme A and NAD are both made from the B vitamins. Cofactors are inorganic substances, usually metal ions. They increase rate of catalysis by binding to the active site of the enzyme so that the enzyme-substrate complex forms more quickly and easily. Examples of cofactors include Cl- in amylase and Zn2+ as a prosthetic group in carbonic anhydrase. Inhibitors Inhibitors are substances that reduce the rate of reaction and fit into a site on the enzyme. Competitive inhibitors Competitive inhibitors have a shape similar to the shape of the substrate and complementary to the shape of the active site. They fit into the active site, stopping the substrate molecules fitting in. This reduces the number of available active sites. The amount of inhibition depends on the relative concentrations of inhibitor and substrate molecules. Non-competitive inhibitors Non-competitive inhibitors fit into a different site (the allosteric site) on the enzyme molecule. They cause a change in the shape of the enzyme molecule. This affects the active site, so the substrate molecule is no longer complementary to the active site and so can no longer fit in. Reversible and non-reversible inhibitors Reversible inhibitors occupy the enzyme site only briefly whereas non-reversible inhibitors bind permanently to the enzyme. Drugs and poisons Many metabolic poisons act by inhibiting enzymes. A poison such as cyanide inhibits the action of the enzyme cytochrome oxidase in aerobic respiration. Alpha amanitin causes death by inhibiting the production of mRNA, preventing transcription and translation and thus protein synthesis. This means vital proteins in the body that are needed for survival like haemoglobin are not produced. Cytochrome oxidase contains iron ions and the cyanide binds to them. Many medicinal drugs also act as inhibitors of enzymes in the body. Aspirin binds to enzymes, preventing the formation of cell-signalling molecules that normally stimulate pain sensitivity. This is why taking the correct dose of medicinal drugs is important as overdosing can be lethal, especially if the inhibitor is non-reversible. Product inhibition Product inhibition occurs when the product of an enzyme-controlled reaction inhibits the enzyme. This can act to prevent too much product being formed.