ENZYMES - structure, function, optimum conditions

and enzyme reaction investigation experiments

See also Enzymes and Biotechnology (gcse chemistry notes)

Doc Brown's Biology Revision Notes

Suitable for GCSE/IGCSE/O level Biology/Science courses or equivalent

 This page will help you answer questions such as ...

 What is the structure of enzymes?

 What is the 'key and lock' mechanism?

 What is the structure of enzymes?

 Why are enzymes most effective in a narrow pH or temperature range?



What are enzymes? and why are they so important in living systems

Enzymes are complex protein molecules that catalyse most chemical reactions that go on in cells.

Every reaction has a specific enzyme that catalyses it - referred to as the specificity of an enzyme.

Enzymes are true biological catalysts, speeding up reactions without being used up in the chemical processes they facilitate.

Every protein an organism requires, including enzymes, is coded for by a different gene in the DNA. This specific gene not only determines the sequence of the amino acids in an enzyme protein, but also its unique shape, and it is the shape that largely determines what an enzyme can do.

The rate of chemical reactions are increased by increase in temperature, but high temperatures may harm the structure and function of complex biological molecules. Therefore the catalytic power of enzymes in speeding up reactions enable organisms to live at relatively low temperatures.

Without them there would be no photosynthesis in plants, protein synthesis and respiration in plants and animals, so without these metabolic processes there would be no life!

e.g. mitochondria contain all the enzymes needed for the chemical reactions involved in respiration - the source of energy to power cells.

Moreover, all the chemical processes of life must well controlled to keep things in balance so any organism can function properly. This involves enzymes and hormones for example e.g. the right levels of sugar in the blood. The appropriate concentrations of substrates and products as well as temperature all important variables that need controlling. If the rates of so many chemical reactions are not controlled in harmony with each other, then cells may be damaged beyond repair hence endangering the whole organism.

See also Enzymes and Biotechnology (gcse chemistry notes)

 


How do enzymes work?

A substrate molecule is a reactant which is to be changed into the product by way of the specific enzyme.

The substrate molecule (or molecules) must fit neatly into the active site on an enzyme and weakly bond to it.

The enzyme, or more specifically, the active site, is referred to as the 'lock', and in an analogy with door locks, the substrate molecules are referred to as the 'key or keys'. This gives to the mechanism by which enzymes function as the 'key and lock' mechanism. This is illustrated below.

It is at the active site the chemical change from substrate to product takes place and its shape is very important.

Many biochemistry reactions either involve synthesis of a larger molecule by joining smaller ones or breaking down and splitting a larger molecule into smaller ones.

The following diagrams illustrate two examples of the 'key and lock' mechanism - how an enzyme works. It is sometimes quoted as a hypothesis, but there is a vast amount of evidence to show this mechanism is correct.

Each enzyme is shaped precisely to accept the substrate molecules, otherwise the reaction will NOT take place, which is why a particular enzyme can only catalyse a specific reaction. The substrate must fit into the active site.

If the enzyme is not the right shape e.g. the protein structure-active site is damaged, the substrate molecule cannot 'key in' so the enzyme cannot function and the reaction does not take place. This protein structure damage is referred to as a denaturing of the enzyme. It can be caused by too high a temperature or the medium may be too acid (too low a pH) or too alkaline (too high a pH) - see later section on factors affecting the rate of enzyme reactions.

(Stage 1) is the 'docking in' of the substrate molecules into the active sites, they are held there sufficiently to allow the chemical transformation to take place.

(Stage 2) happens on the active site where the substrates are catalytically changed to products which are then released from the enzyme.

key and lock mechanism

Sequence key e.g. for a larger molecule being made from two smaller molecules, perhaps a stage in protein synthesis

E = free enzyme (the 'lock'), S = free substrate reactant molecule (the 'keys')

ES = enzyme-reactants complex, EP = enzyme-product complex, E = free enzyme, P = free product

 

key and lock mechanism

Sequence key e.g. for a larger molecule being broken down into two smaller molecules, perhaps in digestion

E = free enzyme (the 'lock'), S = free substrate reactant molecule (the 'key'),

ES = enzyme-reactant complex, EP = enzyme-products complex, E = free enzyme, P = free products

 

See also Enzymes and Biotechnology (gcse chemistry notes)

 


Factors that affect an enzyme's performance

What is the effect of changing concentration for an enzyme catalysed reaction?

(c) doc b

Concentration: If the substrate reactant e.g. sugar, concentration is increased, the rate of reaction increases in a simple proportional way.

Kinetic particle theory: The greater the concentration of substrate (or enzyme, see below) The greater the probability of a fruitful collision leading to the formation of products.

 See Rates of Chemical Reactions Notes

The graph on the left shows what happens as you gradually increase the substrate molecule concentration for a fixed constant concentration of enzyme. Initially the rate of reaction steadily increases, in fact, usually, it rises proportionately with increase in substrate concentration, but ....

What happens if the enzyme gets overloaded with substrate molecules?

However, if the concentration of enzyme is low but the substrate concentration becomes very high, the rate of reaction rises to a maximum and then stays constant. This is because the maximum number of catalyst sites for the 'key and lock' mechanism are all in use and the maximum rate of reaction depends on the rate of diffusion of substrate in and diffusion product out of the active sites. This shows up as the later horizontal portion on the graph - meaning the speed of the reaction has become constant because there are no more active sites to bring into operation.

What happens if you change the enzyme concentration?

At constant substrate concentration, the rate of reaction (usually) increases linearly with increase in enzyme concentration. The greater the concentration of enzyme, the greater the probability of a fruitful collision leading to the formation of products. Hence the initial linear part of the graph.

However, if the enzyme concentration is very high and the substrate concentration low, the rate tails off and becomes constant. This is because there are lots of 'active sites' available, in excess of those needed, and adding more of the enzyme makes no difference.

What is the optimum pH of an enzyme catalysed reaction?

(c) doc bpH effect: The structure of the protein enzyme can depends on how acid or alkaline the reaction medium is, that is, it is pH dependent.

If it is too acid (very low pH) or too alkaline (very high pH), the structure of the protein is changed and it is 'denatured' affecting the shape of the 'active site' and becomes less effective.

The two graphs above illustrate how the rate of reaction varies with pH for many enzymes.

The enzyme catalase breaks down harmful hydrogen peroxide into water and oxygen, with an optimum pH range around pH 7 (top left graph). The enzyme amylase, that breaks down starch into smaller sugar molecules, has an optimum pH range of 4.6 to 5.2 (top right graph). Note that an enzyme is active over a pH range of perhaps several pH units, but beyond this range it is relatively ineffective.

In the optimum pH range, the enzyme catalysis is at its most efficient. In the denaturing process the 'active site' (see 'key and lock' mechanism details above) may be damaged by highly acid or alkaline conditions, and changed in such a way that the enzyme cannot perform its catalytic function on the substrate molecules.

activity of selected enzymes versus pHIf the enzyme does not have the correct 'lock' structure in the protein (the 'active site'), it cannot function efficiently by accepting the 'key' substrate molecule. Most enzymes have an optimum pH of between 4 and 9, and quite frequently near the neutral point of pH 7. Our own body fluids e.g. in blood or cell fluids have a pH of ~pH 7.2 to 7.4, so its no coincidence that many of our enzymes have an optimum operating pH ~7, but it does depend on where you are in your body!

However, the enzyme pepsin has a peak at pH 2 (graph on right) and can operate in the very acid (hydrochloric) conditions of the stomach to help breakdown proteins for digestion in the small intestine.

What is the optimum temperature for an enzyme catalysed reaction?

(c) doc b

Temperature: The structure of the protein enzyme can depend the temperature. If the enzyme does not have the correct 'lock' structure, it cannot function efficiently.  The shape of the graph is due to two factors.

(1) The initial rise in rate of reaction is what you normally expect for any chemical reaction. The increase in temperature increases the average kinetic energy (KE) of the molecules to increase the chance of the product forming from the higher KE 'fruitful collisions. In other words more molecules have sufficient energy to overcome the activation energy to break bonds. There is also an increase in the rate of collision of the substrate and enzyme molecules.

(2) However as the temperature rises further, the increasing thermal vibration of the enzyme molecule causes its structure to break down (denature) and so the 'lock' is damaged so the enzyme is less efficient in interacting with the substrate molecule (see key-lock below). It means the substrate molecule cannot properly dock into the active site on the enzyme.

This may be due to the failure of weak intermolecular forces or actual ionic/covalent bonds, but the 3D molecular structure  of the enzyme is changed so that the substrate molecule cannot 'dock into' the active site on the enzyme to be changed into products. This damage, the denaturing of the enzyme is irreversible. It will not go back to its normal shape even if the reaction mixture is cooled down.

The optimum temperature for the fastest rate of reaction is often around 30-40oC (note our body temperature is about 37oC, no coincidence!). Eventually at high temperatures the enzyme completely ceases to function.

Note: NOT every enzyme reaction has an optimum of ~30-40oC!, some organisms exist and survive at very low temperatures and some at very high temperatures - extremophiles.

See Adaptations, lots of examples explained including extremophiles

(3) The graph that you see is effectively the result of adding two graphs together, (1) the increase in rate due to increase in temperature and (2) the decrease in rate as denaturing of the enzyme increases with increase in temperature. The resulting graph then has two minimums and one maximum, the hump in the graph is the point of maximum speed of the reaction, 'highlighting' the optimum temperature range.

 

 


METHODS OF MEASURING ENZYME ACTIVITY

This section is about ideas and possible experimental procedures for investigating the factors that control the rates of enzyme catalysed chemical reactions. I do not give detailed recipes for the experiments - just lots to think about!


 How might the factors controlling enzyme activity be investigated in the laboratory?

Introduction

You can measure the rate of formation of a product (product/time) or the rate at which the substrate is used up e.g. time taken for a given amount of substrate to react and be used up.

The experiments described below can be adapted to look at the three main factors (variables) which can affect and control the speed of an enzyme catalysed reaction.

Theoretically there are three main variables you can investigate for any enzyme reaction. The concentration of the substrate or the concentration of the enzyme, the temperature and the pH (how acidic/alkaline) of the medium can all affect the speed of an enzyme catalysed reaction.

This means you must control ALL the variables to make the experiment a 'fair test'. All the variables involved in an experimental investigation must be kept constant except for the effect of the variable you are investigating.

(a) Varying the concentration of the substrate or enzyme at a constant temperature (and constant pH) - room temperature being the most convenient. You must keep either the substrate concentration, or the enzyme concentration constant, and monitor the rate effect of changing the concentration of the other.

The laboratory temperature can vary even in a single lesson so using a thermostated water bath is advisable - if available. A temperature of 25oC to 35oC is likely to be suitable.

The pH should be automatically fairly constant and no buffer should be needed unless any acidic or alkaline substances are formed or removed as a consequence of the enzyme catalysed reaction. Your body automatically does this by various chemical means, to keep most of your cell and body fluids around ~pH 7.3, safe!

A buffer is a special solution of chemicals that resists pH changes if small amounts of acid or alkali are added to, or removed from, a solution.

(b) Varying the temperature of the reaction mixture keeping the concentrations of substrate and enzyme constant, this automatically keeps the pH constant in most cases.

Again, using a thermostated water bath is advisable and the best way to vary the temperature e.g. conducting experimental runs at 20oC, 30oC, 40oC etc.

(c) Varying the pH of the reaction medium using buffer solutions. The temperature and concentrations of substrate and enzyme must be kept constant.

Again, using a thermostated water bath is advisable to keep the temperature constant e.g. somewhere in the range 25oC to 35oC.

To get good results takes a lot of hard careful work, but the experiments can be divided out amongst a class ad the pooled data analysed by all the class.

See also GCSE chemistry notes: How can we measure the speed or rate of a chemical reaction?


Method 1. A method of measuring the rate of product formation from an enzyme reaction

In this experiment you are measuring the rate at which oxygen is formed from the enzyme catalase decomposing hydrogen peroxide. Here, the product oxygen gas, provides the means of following the rate of the reaction.

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

The basic procedures for method 1.

(a) Investigating the effect of changing the concentration for an enzyme reaction (hydrogen peroxide to oxygen using catalase)

(either the hydrogen peroxide or the enzyme catalase).

Mashed up potato made into a fine slurry diluted with acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

A slurry is a pulverized solid mixed in a liquid.

You need a series of hydrogen peroxide solutions of known different concentrations and fixed concentration of the potato mix to investigate the effect of changing the hydrogen peroxide concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You can also keep the hydrogen peroxide concentration constant and do the investigation with a set of different concentrations of the potato-catalase mixture.

The water bath is set to a constant temperature e.g. 25oC. The apparatus is setup as illustrated above.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

The 'stock' solutions of potato-catalase or hydrogen peroxide should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just put together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a fixed amount of the same hydrogen peroxide solution into the boiling tube using a pipette or more accurately with a 10 cm3 measuring cylinder. You should keep the total volume of the reaction mixture the same.

(There shouldn't be a need for a buffer, but the mixture should have a constant pH of ~7)

You should make up the reaction mixture of hydrogen peroxide and potato slurry as quickly as possible and shake well.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.

You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(or set of volume readings for one run, and plot graph of volume versus time and measure the initial gradient, but more work for repeats - see graph on the right)

However you get the results, the rate is calculated as follows:

Speed of reaction expressed as:

rate = volume of O2 formed/time taken (cm3/s)

You then repeat the whole exercise with different concentrations of either the hydrogen peroxide or the enzyme, keeping the other one constant.

(c) doc bYou then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus temperature.

You should find that the rate increases with increase in either hydrogen peroxide or enzyme concentration, you may get a nice linear graph like the one on the right or else!

 

Gas syringe system

It is possible to get more accurate results using a gas syringe system, as long as the flask can be set up in a water bath (omitted from the diagram below!) or the laboratory temperature stays constant.

Factors affecting the rates of Reaction - theory and methods of measuring the speed of a reaction (c) Doc Brown

You can get more accurate data of the volume of oxygen formed over time and from the graphs work out the initial rate of reaction from the initial gradient (see right-hand side of above diagram).

Graph line A (steeper gradient) compared to graph line B may represent (i) an increase in concentration of either substrate or enzyme, or (ii) an increase in temperature or (iii) a solution pH nearer the optimum value for that particular enzyme.

See also GCSE chemistry notes: Effect on rate of changing reactant concentration in a solution

 

(b) Investigating the effect of changing temperature for an enzyme reaction (hydrogen peroxide to oxygen using catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You should also use the same volume of the well shaken potato slurry.

The water bath is set to the start temperature e.g. 20oC. The apparatus setup is illustrated above.

You could start as low as 10oC perhaps by cooling the water with ice, not sure how well it would work?

The two 'stock' solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature  - solutions and boiling tube.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a fixed amount of the same hydrogen peroxide solution into the boiling tube using a pipette or more accurately with a 10 cm3 measuring cylinder. You should keep the total volume of reaction mixture constant.

(There shouldn't be a need for a buffer, but the mixture should have a constant pH of ~7)

You make up the reaction mixtures as quickly as possible in a boiling tube and shake well.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.

You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

Repeats will allow a more accurate average value of the rate of reaction to be used in the final analysis.

  (or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work for repeats - see the graph on the right)

Speed of reaction expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise at 30oC, 40oC, 50oC etc. adjusting the thermostat temperature control.

You should find from 20oC to 40oC an increase in the rate of oxygen production, but an increasingly slower rate of reaction from 50oC to 70oC (see graph on bottom right).

You then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus temperature.

See the end of method 1. (a) for a gas syringe method

See also GCSE chemistry notes: Effect on rate of changing the temperature of reactants

 

(c) Investigating the effect of changing pH for an enzyme reaction (hydrogen peroxide to oxygen using catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration AND stock solution of the potato slurry to provide the enzyme catalase.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You need stock solutions of buffers giving a variety of pH values.

The water bath is set to a constant temperature e.g. 25oC. The apparatus setup is illustrated above.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - measure and monitor.

The 'stock' solutions of catalase, hydrogen peroxide and the buffer solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amounts of the potato slurry, hydrogen peroxide solution and the buffer solutions into the boiling tube using a pipette or more accurately with a 10 cm3 measuring cylinder.

Whatever your 'recipe', keep the total volume of the three solutions constant for the final reaction mixture.

The three solutions are mixed in a boiling tube and well mixed, the total volume should be constant and you use the same concentrations of the hydrogen peroxide and potato-catalase. The pH of the buffer should be the only variable.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.

You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work doing repeats - see the graph on the right)

Speed of reaction expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise with different pH buffer solutions.

You then draw a graph of the mean values of the rate of reaction (in cm3/s) versus the pH, and it 'may' look like the graph on the right.

See the end of method 1. (a) for a gas syringe method

 


Method 2. A method of measuring the reduction of substrate concentration in an enzyme reaction

In this case you are detecting when the substrate (starch) is used up when it is broken down to maltose by the enzyme amylase.

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

2(C6H10O5)n  + H2O  ====> nC12H22O11

Starch is a natural polymer where n is a very large number.

This reaction is called a 'hydrolysis' because a molecule reacts with water to give two or more products.

You can use 'baby rice' for this experiment!

The method for this particular reaction relies on detecting the presence of starch using iodine (illustrated above). You use a dilute solution of iodine dissolved in potassium iodide solution, which is orangey-brown in colour. When added to a starch solution a blue-black colour is seen. This is a simple standard biological laboratory test for starch and you simply take a sample from the reaction mixture and test it for starch using the iodine solution in the wells of a spotting tile. When you no longer see a blue-black colour, all the starch is used up and you note the time taken.

This method is fine in a school/college laboratory where simple apparatus will be available for several groups in a class. However it is possible to measure the intensity of the blue-black colour using an electronic instrument called a colorimeter. A sample of the reaction mixture is put into a special tube and the colorimeter apparatus can measure the intensity of the blue-black colour. A special light filter allows you to measure just the intensity of the colour you are interested in. You also take a blank reading without the starch present. From a calibration graph of known starch concentrations versus their colour intensity with iodine you can actually measure the concentration of the starch as it decreases with time. This method does avoid the uncertainty of when the blue-black colour actually completely disappears. In the methods described below you are measuring the average rate and the rate is varying all the time. This colorimeter method avoids errors due to the varying speed of the reaction, which is always slowing down!

Light from a suitable source is passed through a light filter to select the most appropriate wavelength of light, some of which is then absorbed by the solution held in a special glass cuvet (a sort of 'test tube'). The amount of light absorbed is called, and measured as, the absorbance which is a function of the coloured solute concentration.

 

The basic procedures for method 2.

(a) The effect of changing concentration for an enzyme reaction (starch to maltose using amylase)

(either the starch or amylase)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

The theory of the method is explained at the start of the method 2. section

You can start with 1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The thermostated water bath should be set to a suitable temperature e.g. 25oC.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

The boiling tubes of stock solutions of starch and amylase of varying concentrations should be put into the water bath, so everything involved is at the same start temperature.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile with a pipette.

Portions of the starch and amylase are carefully measured out into a boiling tube and shaken well and immediately placed in the water bath and the stop watch started.

With a clean teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch. Eventually the iodine solution is unchanged and you note the total time elapsed.

Repeat several times so you can calculate a more accurate average reaction time for the final analysis.

You then repeat the whole exercise with different concentrations of starch, keeping the amylase concentration constant OR varying concentrations of amylase and keeping the starch concentration constant.

(c) doc bThe reciprocal of the time (1/time) for the starch to be used up gives you a measure of the rate of the enzyme reaction.

As the concentration of either starch or enzyme is increased, the reaction time should decrease - upper graph on the right.

(c) doc bYou then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus concentration - lower graph on the right. The graph should show an optimum value of the maximum rate of reaction for that particular mixture.

See also GCSE chemistry notes: Effect on rate of changing reactant concentration in a solution

 

(b) The effect of changing temperature for an enzyme reaction (starch to maltose using amylase)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

The theory of the method is explained at the start of the method 2. section

1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The thermostated water bath should be set to an initial temperature e.g. 20oC.

The boiling tubes of stock solutions of starch and amylase should be put into the water bath, so everything involved is at the same start temperature.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile.

Portions of the starch and amylase are carefully measured out into a boiling tube which is immediately placed in the water bath and start the stop watch.

With a teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch. Eventually the iodine solution is unchanged and you note the total time elapsed.

Repeat several times so you can calculate a more accurate mean reaction time for the final analysis.

You then repeat the whole exercise at 30oC, 40oC, 50oC etc. adjusting the thermostat temperature control.

The reciprocal of the time (1/time) for the starch to be used up gives you a measure of the rate of the enzyme reaction.

(c) doc bYou should find from 20oC to 40oC a decrease in total reaction time for the starch to be used up, but an increasingly longer reaction time from 50oC to 70oC.

You then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus temperature. The graph should show an optimum value of the maximum rate of reaction for that particular mixture.

See also GCSE chemistry notes: Effect on rate of changing the temperature of reactants

 

(c) The effect of changing pH for an enzyme reaction (starch to maltose using amylase)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

The theory of the method is explained at the start of the method 2. section

1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

You need a good range of buffer solutions, preferably from pH 2 to pH 10.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The thermostated water bath should be set to a suitable temperature e.g. 25oC.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

The boiling tubes of stock solutions of starch, amylase and various buffer solutions of differing pH should be put into the water bath, so everything involved is at the same start temperature.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile.

Portions of the starch, amylase and buffer solution are carefully measured out into a boiling tube which is immediately placed in the water bath and start the stop watch. You should keep the total volume of the final mixture constant.

With a teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch. Eventually the iodine solution is unchanged and you note the total time elapsed.

Repeat several times so you can calculate a more accurate average reaction time for the final analysis.

 You then repeat the whole exercise with buffers of differing pH.

The reciprocal of the time (1/time) for the starch to be used up gives you a measure of the rate of the enzyme reaction.

You then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus the pH of the buffer solution.

You should get a peak around pH 5, a bit like the graph on the right.

 


See also Enzymes and Biotechnology (gcse chemistry notes)


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