enzymes (as level bio)

7/24/2019 Enzymes (AS Level Bio)

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By; Dr. Mufaddal Page 1

nzymes

Active Site, Activation Energy & Enzyme Specificity

Enzymesare globular proteinsthat serve as biological catalysts.

They speed up or slow down metabolic reaction, but remain unchanged.

They may facilitate the breaking of an existing bond or the formation of anew bond.

Substrates= the molecules that bind to the enzymeProducts = new substances formed.

1. Active Sites

Active site= area in enzyme's molecule where the substrate bind to

enzyme --> enzyme-substrate complex.

The R groupsof amino acids at the active site form temporary bonds withthe substrate molecule. This pulls the substrate slightly out of shape, causing it

to react and form products.


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2. Activation energy

Activation energy= energy the substrates need for changing themselves

into products. Heatingprovides activation energy.

Enzymes reduce activation energy needed ---> reaction take place

at low to. They do this by distort the shapeof the substrate when it binds

at the enzyme's active site.


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3. Enzyme specificity

Lock and Key hypothesis(Emil Fisher, 1894)

The shape of the active siteof the enzyme and the substratemolecules

are complementary.

They possess specific 3-D shapes that fitexactlyinto one another.

Like a key into a lock, only the correct size and shape of the substrate (the

key) would fit into the active site of the enzyme (the lock).

This shows the high specificity of enzymes, however it is too rigid.

The active site has a complementary shape to the substrate.

Induced fit hypothesis (Koshland, 1958)

The shape of the active siteof the enzyme and the substratemolecules

are NOT complementary.

In the presence of the substrate, the active site continually reshapesby its

interactions with the substrate, until the substrate is completely fit into it.

The enzyme is flexible and molds to fit the substrate molecule like glovesfitting ones hand or clothing on a person.

This hypothesis is more acceptable.


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The active site forms a complementary shape to the substrate only after

binding.

Following the course of an enzyme-catalyzed reaction

Measurement of the rateof formationof the product or the rate ofdisappearanceof the substrate.

1. Measurement of the rate of formation of O2in the reaction:

Mash up some biological material like potato tuber or celery stalks, mix them

with water and filter the mixture to obtain a solution containing catalases. Add the mixture to H2O2(hydrogen peroxide) in a test tube.

Use small tubes --> not too much gas in the tube above the liquid.

Collect the gas in a gas syringe and recording the volume every minute untilthe reaction stops.

Note - You can replace the gas syringe by an inverted measuring cylinder overwater.


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2. Measurement of the rate of disappearance of starch in the reaction:


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Add amylasesolution to starchsuspension in a test-tube.

Take samples of the reacting mixture at regular time intervals, and test forthe presence of starch using iodinein KI solution.

Whenstarch is present, iodine is dark blue.

If the blue color lightens, starch is breaking down.

When there is no starch, the iodine solution will remain orange-brown.

To obtain quantitative results, use a colorimeter.

Put some of the iodine solution into one of the colorimeter tubes, place it in

the colorimeter and adjust the dial to give a reading of 0. This is yourstandard, with no starch.

Every minute, take a sample of the liquid from the starch-amylase mixture

and add it to a clean colorimeter tube containing iodine solution. Mixthoroughly, and then measure the light absorbance.

The darker the blue-black color, the greater the absorbance, and the greaterthe concentration of starch.


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Factors affecting the rate of enzyme-catalyzed reactions

These factors are:

- Temperature

- pH- Enzyme concentration

- Substrate concentration

- Inhibitor concentration

When an enzymesolution is added to a solution of its substrate, the

molecules collide.

With time, the quantity of substrate (changed into product) --> frequency of

collisions --> rate of reaction gradually . The reaction rate is fastest at the

start of the reaction (substrate concentration is greatest). --> When comparingreaction rates of an enzyme in different circumstances, we should measure

the initial rate of reaction.

1. Temperature

As to, kinetic energy of reacting molecules --> successful collision --> rate of reaction.

At optimal to enzyme's activity is maximal --> rate is maximal.

Above this temperature, H bonds holding enzyme molecule in shape begin tobreak --> change tertiary structure of the enzyme (denaturation) --> activesite is deformed ---> binding of substrate with enzyme --> rate of

reaction.


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Enzymes in the human body generally have an optimum to of about 37oC,

organisms that have evolved to live in much higher or lower temperatures may

have much higher or lower optimum to.

2. pH

Most enzyme molecules only maintain their correct tertiary structure (exhibit

maximum activity) within a very narrow pH range.

Optimum pH- is the pH at which an enzyme has maximum activity.

Biological buffers help maintain the optimum pH for an enzyme.

Changes in pH can make and break intra- and intermolecular bonds,changing the shape of the enzyme and, therefore, its effectiveness.

Most enzymes have an optimum pH that falls within the physiological rangeof 7.0-7.5.

Notable exceptions are the digestive enzymes pepsin and trysin:pepsin(active in the stomach) - optimum pH of 1.5

trypsin(active in the small intestine) - optimum pH of 8.0.


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3. Enzyme concentration

When there are more substrate than enzyme:

Limiting factor isEnzyme concentration

concentration of enzyme --> collisions between enzyme and substrate -->rate of the reaction (directly proportional to the enzyme concentration )

Increasing the enzyme concentration beyond a certain pointdoes not

change the rate of reaction BECAUSE when there are less substrate than

enzyme:

limiting factor isSubstrate concentration

Concentration of enzyme does NOTrate of reaction.

Limiting factor:

Factor that directly affects the rate of reaction at which a process occurs if itsquantity is changed.

Its value has to be in order to the rate.

4. Substrate concentration

When there is more enzyme than substrate:

Limiting factor is Substrate concentration

concentration of substrate --> collisions between enzyme andsubstrate -->rate of the reaction

Increasing the substrate concentration beyond a certain pointdoes not

change the rate of reaction BECAUSE when there are less enzyme than

substrate:

limiting factor isEnzyme concentration

Concentration of substrate does NOTrate of reaction.


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5. Inhibitor concentration

Inhibitor = a substance that slows down the rate at which an enzyme works.

Competitive inhibitors

Have similarshape to the enzyme's normal substrate.

Canfit into the enzyme's active site, preventing the substrate frombinding.

The greater the proportioninhibitor: substrate, the more inhibitormolecules (not substrate molecules) will bump into an active site.

Relative concentrationsof the inhibitor and the substrate will affectthedegree to which a competitive inhibitor slows down a reaction.

Non-competitive inhibitors Have different shape than the substrate.

Do notbindto the active site.

Bind to a different partof the enzyme --> changes the enzyme's shape(including the active site) --> substrate cannot bind with enzyme.

A relative concentration of the inhibitor and the substrate doesnotaffectthe degree to which a non-competitive inhibitor slows down a reaction

(if you add more substrate, it still won't be able to bind).


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Investigating factors affecting the rate of enzyme-catalyzed reactions

1. Temperature

The effect of toon enzyme activity

You can use almost any enzyme reaction for this, such as the

action of catalase onH2O2. You could use the same method of collecting the

gas that is described there, but here is another possible method:

Set up several conical flaskscontaining H2O2 (the same volumeand concentration). Stand each one in a water bath at a particular to. Use at

least 5 toover a good range (0-90oC). Better to set up 3 sets of tubes ateach to --> mean result for each to.

Take a set of test tubesand add the same volume of catalasesolution to

each one. Stand these in the same set of water baths.

Leave all the flasks and tubes to come to the correct to. Check with athermometer.

Take the first flask with H2O2, dry its base and sides and stand it on a

sensitive top-pan balance. Pour in the catalase solution (same to) andimmediately take the balance reading.

Record the new balance readings every 30 seconds for about 3 minutes. Thereadings will as O2is given off.

Repeat with the solutions kept at other temperatures.

Work out the initial rate of each reaction, either taken directly from yourreadings, or by drawing a graph of mass lost (mass of O2) against time foreach to, and then working out the gradient of the graph over the first 30seconds or 60 seconds of the reaction.

Use your results to plot a graph of initial rate of reaction (y-axis) against to.


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2. pH

The effect of pH on enzyme activity

You can adapt the method described earlier for investigating the effectof toon the rate of breakdown of H2O2by catalase.

Vary pH by using different buffer solutions added to each enzyme solution.

Keep to, enzyme concentration, substrate concentration and total volume ofreactants the same for all the tubes.

Record, process and display results.

3. Enzyme concentration

The effect of enzyme concentration on rate of reaction

You could use the following method to investigate the effect of enzymeconcentration on the rate at which the enzyme catalaseconverts

its substrate H2O2to H2Oand O2.

Prepare a catalase solution as described earlier.

Prepare different dilutions of this solution:


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And so on. The final 'solution' prepared should be 10 cm3 of distilled

water.

Place each solution into a tube fitted with a gas syringe. Use small tubes -->there is not too much gas in the tube above the liquid, but leave space toadd an equal volume of H2O2solution at the next step. Label tube with awaterproof marker. Better to prepare 3 sets of these solutions.

Place each tube in a water bath at 30oC.

Take another set of tubes and add 10 cm3 of H2O2solution to each one. Theconcentration of H2O2must be the same in each tube. Stand these tubes inthe same water bath.

Leave all the tubes for 5 minutes --> correct to. Add the contents of 1 ofthe H2O2tubes to the first enzyme tube. Mix thoroughly.

Measure the volume of gas collected in the gas syringe after 2 minutes. Ifyou are using 3 sets, then repeat using the other 2 tubes containing thesame concentration of enzyme.

Do the same for each of the tubes of enzyme. Record the mean volumeof gas produced in 2 minutes for each enzyme concentration and plot a

line graph to display your results.

Note:if you find that you get measurable volumes of gas sooner than

2 minutes after mixing the enzyme and substrate --> take readings earlier.The closer to the start of the reaction you make the measurements, the

better.

4. Substrate concentration

The effect of substrate concentration on enzyme activity

You can do this in the same way as described for investigating the effect

of enzyme concentration, but this time keep the concentration of catalase

the same and vary the concentration of hydrogen peroxide.


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Michaelis - Menten Equation and Immobilizing an enzyme

Michaelis-Menten equation describes the velocity of enzymatic reactions

(v) by relating it to [S] - concentration of a substrateS.

Michaelis - Menten Equation

An example curve with parameters

Vmax= 3.4 and Km= 0.4

Here, Vmaxrepresents the maximum rate achieved by the system, at maximum

(saturating) substrate concentrations.

The Michaelis constant Kmis the substrate concentration at which the reaction

rate is half of Vmax.

Kmis (roughly) an inversemeasure of the affinityor strength of binding

between the enzyme and its substrate. The lower the Km, the greater theaffinity (so the lower the concentration of substrate needed to achieve a given

rate).

It permits prediction of whether or not the rate of formation of product will be

affected by the availability of substrate.


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Immobilizing an enzyme in alginate

Inindustry enzymes are used on a large scale.

It is very costly to use enzymes only once, but most enzymes are only

commercially available in liquid or dehydrated forms and once they have been

used in solution it is very difficult and time consuming to separatethem from

the product.

To allow theirre-use, enzymes may be immobilized (attached to an inert,

insoluble material such as calcium alginate to form a gel capsule around

them). This way, the enzymes will be hold in place throughout the reaction,

easily separated from the products and may be used again.

One way of immobilizing enzymes:

mixing theenzymewithsodiumalginate,then dropping the mixtureintocalcium chloridesolution. Sodium alginate will turn from liquid to solid whenimmersed in calcium chloride. This produces small beads with the enzyme inside.

2 Na (Alginate) + Ca++-------> Ca (Alginate)2+ 2 Na+

The substrate that the enzyme acts upon is able to diffuse through the gel,

although this may be quite slow.

Immobilized enzymes are widely used in industry because it allows the

reaction to flow continuously and the product will not be contaminated with the

enzyme so will not need to be purified.


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Summary of Enzymes

1. Enzymes are globular proteins which catalyze metabolic reactions.

2. Each enzyme has an active site with a specific shape, into which the

substrate molecule or molecules fit precisely. This is the lock and key

hypothesis the substrate is compared with a key which fits precisely into

the lock of the enzyme.

3. The lock and key hypothesis has been modified. The modern hypothesis is

called the induced fit hypothesis. The active site is no longer regarded as a

rigid structure like a lock, but as a flexible structure which can change shape

slightly to fit precisely the substrate molecule.


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4. When the substrate enters the active site, an enzymesubstrate complex is

temporarily formed in which the R groups of the amino acids in the enzyme

hold the substrate in place.

5. Enzymes may be involved in reactions which break down molecules or join

molecules together.

6.

Enzymes work by lowering the activation energy of the reactions they

catalyze.

7. The course of an enzyme reaction can be followed by measuring the rate at

which a product is formed or the rate at which a substrate disappears. A

progress curve, with time on the x-axis, can be plotted. The curve is

steepest at the beginning of the reaction, when substrate concentration is at

its highest. This rate is called the initial rate of reaction.

8. Various factors affect the rate of activity of enzymes. Four important factors

are enzyme concentration, substrate concentration, temperature and pH.

9.

The greater the concentration of the enzyme, the faster the rate of reaction,provided there are enough substrate molecules present. Similarly, the

greater the concentration of the substrate, the faster the rate of reaction,

provided enough enzyme molecules are present. During enzyme reactions,

rates slow down as substrate molecules are used up.

10. Each enzyme has an optimum temperature at which it works fastest. As

temperature increases above the optimum temperature, the enzyme

gradually denatures (loses its precise tertiary structure). When it is

completely denatured, it ceases to function. Denaturation is sometimes

reversible.

11.

Each enzyme has an optimum pH. Some enzymes operate within a

narrow pH range; some have a broad pH range.

12. Enzymes are also affected by the presence of inhibitors, which slow down

their rate of reaction or stop it completely.

13. Competitive inhibitors are molecules which are similar in shape to the

normal substrate molecules. They compete for the active site of the enzyme.

This type of inhibition is reversible because the inhibitor can enter and leave

the active site.

14. Non-competitive inhibitors either bind permanently to the active site or

bind at a site elsewhere on the enzyme, causing a change in shape of theactive site. Such binding may or may not be reversible.


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MCQs

1. Which of the following describes an enzyme?

A a catalyst with an active site which binds to the product of a reaction

B a fibrous protein with an active site which binds to a substrate

C a globular protein with hydrophilic groups on its surface

D an insoluble biological catalyst

2. The graph shows the energy changes during the progress of a chemical

reaction.

Which of the energy changes could be decreased by adding an enzyme?

A 1, 2 and 3

B 1 and 2 only

C 1 and 3 only

D 2 and 3 only

3. Which tests, carried out on samples taken at intervals during the course of

the reaction below, would enable the progress of the reaction to be followed?


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1 Benedicts test

2 Biuret test

3 Emulsion test

4 Iodine in potassium iodide solution test

A 1 or 2

B 1 or 4

C 2 or 3

D 3 or 4

4.Which of the following describes the effects of temperature on an enzyme-

controlled reaction?

A At low temperatures, substrate molecules only rarely collide with an

enzymes active site.B At low temperatures, the enzyme loses its shape and activity.

C At low temperatures, the enzyme becomes denatured.

D As the temperature increases, the number of collisions between enzyme and

substrate decrease.

5.Which statement does not describe the effect on an enzymes activity of

changing the pH?

A A pH that is very different from the enzymes optimum pH can denature the

enzyme.

B At low pH there are fewer hydrogen ions to interact with the R groups of the

amino acids that make up the enzyme.

C Changing the pH alters the interactions of the amino acids that make up the

enzyme.

D Changing the pH alters the enzymes three-dimensional shape.

6. The graph shows the effect of increasing the substrate concentration on the

rate of an enzyme-catalyzed reaction.


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What explains why the rate of reaction levels off?

A All the enzyme active sites are saturated with substrate.

B All the substrate has been used up.

C The concentration of the enzyme is gradually decreasing.

D The rate at which enzyme and substrate collide is decreasing.

7. Curve X shows the progress of an enzyme-catalyzed reaction under

optimum conditions. Curve Y shows the same reaction with one factor changed

throughout the reaction.

Which factor was changed?

A A competitive inhibitor was added.

B A non-competitive inhibitor was added.

C The reaction took place at a different temperature.

D The reaction took place at a different pH.


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8.These statements are about enzyme inhibitors.

1 binds reversibly to an enzyme

2 binds to a site on the enzyme different from the active site

3 has a structural similarity to the enzymes normal substrate

4 can be displaced from the enzyme by a high concentration of the enzymes

normal substrate

Which statements describe a competitive inhibitor?

A 1, 2, 3 and 4

B 1, 3 and 4 only

C 2 and 3 only

D 3 and 4 only

9.Fibrous proteins from dead cells is difficult to remove from contact lenses.

Some cleaning solutions contain an enzyme to digest this protein to soluble

products.

What describes the enzyme and its activity?

A An active site on a fibrous protein binds to the enzyme and is hydrolyzed.

B An active site on a soluble product binds to the enzyme and is digested.

C An active site on a globular protein binds to a soluble product and digests it.

D An active site on a globular protein binds to a fibrous protein and hydrolyses

it.

10.Several different organisms produce lactase enzymes to hydrolyze lactose.

The different enzymes have different molecular sizes.

Which description of these different lactases is correct?

A Their active sites have the same shape.

B Their primary structure is the same.C They each contain the same number of amino acids.

D They have exactly the same three-dimensional structure.


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By; Dr Mufaddal Page 23

Answer for MCQs :

1 C

2 C

3 B

4 A

5 B

6 A

7 B

8 B

9 D

10 A

enzymes (as level bio)

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