Investigate the effect of sucrase concentration on the rate of hydrolysis of sucrose.

Andrew Mackay 12 E As Biology Coursework 2002

Skill P – Planning

Hypothesis – The digestion of sucrose into glucose and fructose is carried out by the presence of the enzyme Sucrase. The activity of the enzyme will be greatest at its optimum temperature since at this temperature the enzyme’s tertiary structure will be correct to allow the formation of the substrate-enzyme complex quickly and efficiently. This should be at 37°C (body temperature), which is the temperature of the ileum where Sucrase is secreted and naturally functions.

The first thing to say about enzymes is that they are proteins and they are found in all types of organisms from humans to viruses. They function in the body as catalysts. In other words they speed up the rate of chemical reactions in the body. Enzymes actually accelerate chemical reactions by a factor of about 1 million. Enzymes are organic catalysts. Without enzymes the metabolism of an organism would be too slow for the organism to survive. The word enzyme actually means "in yeast" as they were first discovered in these micro-organisms. Enzymes are found inside cells (intracellular) and outside cells (extra cellular). Intracellular enzymes are for example, those that control metabolism while extra cellular enzymes are for example, gut digestive juices and the enzymes secreted by bacteria, which digest their food outside the body then reabsorb the products. Enzymes work on particular organic chemicals, these are called substrates. So in effect food is the substrate of digestive enzymes. Each enzyme is specific to one or a group of particular substrates. The majority of the reactions that occur in living organisms are enzyme-controlled. Without enzymes toxins would soon build up and the supply of respiratory substrate would decrease. Enzymes are proteins and thus have a specific shape. They are therefore specific in the reactions that they catalyse - one enzyme will react with molecules of one substrate. The site of the reaction occurs in an area on the surface of the protein called the active site. Since the active site for all molecules of one enzyme will be made up of the same arrangement of amino acids, it has a highly specific shape. Generally, there is only one active site on each enzyme molecule and only one type of substrate molecule will fit into it. Lock and key hypothesis description of the specificity of the shape of the active site for a particular substrate. The lock and key hypothesis attempts to explain how enzymes are specific to particular substrates and how they may work. In this hypothesis the enzyme is the lock and the substrate(s) is the key. Enzyme molecules have a particular shape like a lock and only a particular substrate (key) can fit into that lock. The part of the enzyme that binds the substrate is called the "active site". Just like other catalysts, after the enzyme has been involved in the reaction it is not altered. Enzymes can be involved in reactions that synthesise products, these are called anabolic reactions or they can be involved in reactions that breakdown products, these are called catabolic reactions. It is these anabolic and catabolic reactions that constitute an organism’s metabolism. Most enzymes are proteins which act as catalysts, regulating biochemical reactions that occur in cells. The molecule that an enzyme acts on is called a substrate and when the two combine they are referred to as an enzyme–substrate complex. The catalysts of biochemical reactions accelerate the rate of reactions that would normally take place. The enzyme acts by stressing the chemical bonds of a substrate thereby lowering the energy required to form new bonds.

The enzyme is not changed by the reaction.

Only small amounts of enzyme are needed in a reaction.

The enzyme can be used again.

Enzymes are specific to a particular reaction.

Most enzymes are globular proteins with one or more active sites. The active site is a region within the enzyme molecule to which the substrate binds. The stress being applied to the substrate, induced to fit into the enzyme, causes the bonds to break in the substrate. The induced fit model of enzyme reactions is the current model being used to demonstrate the enzyme–substrate complex. The earlier ...

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The enzyme is not changed by the reaction.

Only small amounts of enzyme are needed in a reaction.

The enzyme can be used again.

Enzymes are specific to a particular reaction.

Most enzymes are globular proteins with one or more active sites. The active site is a region within the enzyme molecule to which the substrate binds. The stress being applied to the substrate, induced to fit into the enzyme, causes the bonds to break in the substrate. The induced fit model of enzyme reactions is the current model being used to demonstrate the enzyme–substrate complex. The earlier model used was called the lock and key model of binding a substrate to its enzyme. It is no longer used to explain the action of enzymes.

Variables

In order to create a fair test, I considered the variables that affect the activity of the enzyme, sucrase. Any variables that were likely to disrupt the results were controlled, and the variables being investigated were varied accurately so that their effect could be measured precisely.

Substrate Concentration – When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes’ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate any quicker. . Substrate concentration: at a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases. Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed.

Temperature:

Enzymes work best at an optimum temperature. Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too. Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work reactions proceed because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going, (the reaction is not spontaneous). The energy required to initiate the reaction is called the activation energy. When the substrate(s) react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrates or the product. Outside the body, high temperatures often supply the energy required for a reaction. This clearly would be hazardous inside the body though! Fortunately we have enzymes that provide an alternative way with a different transition state and lower activation energy. The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sits available. Temperature all enzymes require an optimum temperature at which to work efficiently.

The rate of a reaction will increase with an increase in temperature until the optimum temperature is reached. At temperatures greater than optimum, the kinetic movement of molecules in the enzyme are too great to hold it together, eventually denaturing the enzyme. These two different enzyme reactions show how temperature will affect each differently.

Lock and key theory and induced fit model

An enzyme molecule is globular and very large but only a small part of it, the active site, is involved in the reaction. This animation shows how the shape of the active site matches that of the substrate molecule. The substrate molecule fits into the active site and is held there until the reaction is complete. The product is then released and the enzyme is once again ready to take part in the reaction. This is known as the lock and key hypothesis. The active site has a distinct shape, rather like a lock. Just as only the right 'key' will fit a lock, so only the right substrate has the right shape to fit into the active site.

Induced-Fit Model of Enzyme catalysis

Metabolism is defined as all the chemical processes inside the cells of a living organism. These could be for building up, maintenance or breaking down of living tissue. Reactions in cells tend to occur spontaneously to produce an end product .The reactants are stable before the reactions take place and must be destabilised by breaking bonds in the reactants. The extra energy needed to destabilise the bonds to initiate a chemical reaction is called activation energy. If a chemical reaction can have its activation energy lowered in order to start it off, then the reaction will proceed more quickly. A catalyst will perform this task and in many cases in metabolic processes the catalysts are proteins called enzymes.

Enzymes are held together by ionic bonds which are sensitive to the H+ concentration of the fluid in which the enzyme is dissolved. Most enzymes have an optimum pH level at which they function best.

The diagram below shows an enzyme working optimally at pH 7.

Inhibitors some active sites can be filled by molecules other than substrate. This competitive inhibitor will not enable the substrate to bind to the enzyme. Sometimes the enzyme is mis-shapen by an inhibitor that attaches to the enzyme in non-competitive inhibition. If the active site is changed, the substrate will not fit.

Coenzymes and Cofactors many enzymes require additional compounds in order for them to work. These cofactors act on the active site of the enzyme to facilitate the action of the enzyme. Many of these cofactors are minerals or ions. Cofactors which are nonprotein organic molecules such as vitamins are known as coenzymes. Some coenzymes join with the enzyme, temporarily changing the enzyme's active site enabling the substrate to fit. In all biochemical pathways, the pH, temperature, substrate concentrations and enzyme concentrations must be close to optimal, and cofactors and coenzymes all must be available in order to produce the end product

The experiment I propose is one aimed at determining the rate of a reaction dependant on the concentration of the solution it is in. My question that I will study is this. “Does the changing of the concentration of the sucrose/sucrase relationship have an effect on the rate of the reaction”? To undertake this question I will perform an experiment where different concentrations of sucrase are mixed with set amounts of sucrose and record the time taken for a measurable reaction to take place. some important definitions are:

Enzyme: a protein substance found in living cells that brings about chemical changes; necessary for digestion of food. This is a biological catalyst, a catalyst is defined as; A substance that increases the rate of a chemical reaction by reducing the activation energy, but which is left unchanged by the reaction. The sucrase solution is dependant on the substrate sucrose, a substrate is defined as; the reactant in any enzyme-catalysed reaction. Catalyst

The apparatus for this experiment are listed below.

This is my method for the experiment:

Take and mix the concentrations of sucrase and distilled water as shown below.
Cut up 6 clinistick strips.
Measure out 5ml of sucrose six times.
Heat up water bath to approximately 40oC.
Put all 12 boiling tubes into the water bath.
Heat the tubes up to 40oC.
Mix solutions one by one.
Measure change in preliminary colour and final colour change.
Record data.
Repeat experiment 3 times to create average and make experiment a fair test.

I will experiment using the following ranges:

Before undertaking this experiment I have performed a preliminary test. I used the same concentrations but as soon as the solutions came into contact the Clinisticks went in. the results produced were very erratic and unreliable, the results are on my results sheet. We reviewed our method and decided that the best idea would be to mix the solutions for 10 seconds before adding the Clinisticks this produced much more reliable results. To cut the Clinisticks we use scissors and there is a safety risk of cutting yourself so it is important to be safe with the scissors. As well as this the water baths are at 40oC and there is a small risk of burning fingers.

The reason for the 40oC water baths is to raise the temperature of the solutions as this will bring the solutions closer to the activation energy level and this will mean there is no energy wasted in starting the reaction and so all the results will be more reliable. As well as this the temperature will also make the results accurate as all the solutions are at a constant temperature and as shown above temperature will have a massive effect on the results over just a 5oC range so to avoid such discrepancies the temperature is checked before every experiment to aim for 40oC to commence the reactions.

Before I began this final experiment and finalised this method I went through a few preliminary experiments. The plans, results and conclusions are briefly listed below:

Preliminary 1

Aim : to determine concentration range.

Method : mix 0 –5% concentrations together and time the rate of change of colour on a clinistick.

Data discovered : the 3%+ concentration were very quick and too quick to record reliably. Below 3% the reaction time was recordable and reliable.

Result : I decided to use 0 – 2.5% as it was a wide enough range for an average and was easily measurable.

Preliminary 2

Aim : to determine first and final colour change

Method : from my first preliminary experiment I learnt that it was difficult to determine when the colour had changed and so my results were random to a point. To avoid this I used a colour chart and chose an exact colour that was just different enough from the starting colour. As well as this the final colour was defined.

Results : my results from then on were much more reliable and the average was easier to plot on a graph drawing my expected curve. The first colour change was a loss of the base pink colour and the final colour change is a measurable blue.

Throughout all these preliminary experiments and the final one all laboratory safety precautions will be observed. To check the reliability of my results I will take a mean average of all 3 experiments I finally did. Using the mean of all the experiments I then will work out the standard deviation of the primary colour change and the final colour change.

On the graphs the X axis is numbered 1-6, this corresponds to the Sucrase Concentration 0.0%-2.5%.

Below is the preliminary experiment graph for primary colour change at 0-5% solution.

The line slopes down as the reaction time decreases in relation to the concentration. This was the graph of the preliminary experiment after making the changes due to evidence from preliminary experiment 1 and 2.

Prediction: I predict that the higher the concentration the faster the reaction due to induced fit and lock and key hypothesis.