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1.4Biological Molecules

Proteins- Enzymes — AQA A Level Biology 7402 Revision Notes & Topical Questions

15 min read 26 parts Lesson 17 of 49 Unit quiz

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Enzymes: Active Sites, Specificity and Induced Fit — AQA A LEVEL BIOLOGY 7402 notes: Explain how enzymes lower activation energy, describe enzyme specificity and the induced-fit model and outline the practical to measure activity.Many proteins are enzymes.

  • enzyme-substrate complex
  • induced-fit hypothesis
  • Worked example 1
  • Worked example 2
  • Worked example 3
  • Saying enzymes are used up in the reaction

Section 1 of 26Enzymes as biological catalysts

Many proteins are enzymes. They are 'biological' because they function in living systems and 'catalysts' because they speed up chemical reactions without being used up or changed. Critical to function is the active site, the region where the substrate binds.

Section 2 of 26Specificity and the enzyme-substrate complex

An enzyme has an active site with a specific shape that fits a specific substrate (the shapes are complementary), forming an enzyme-substrate complex.

1
Substrates collide with the active site and this must happen at the correct orientation and speed for a reaction.
2
The shape of the active site depends on the tertiary structure of the protein.
3
For example, catalase binds hydrogen peroxide because they are complementary in shape, whereas DNA polymerase does not.
4
The complex forms only temporarily: the enzyme catalyses the reaction and the product(s) are released.
5
Both the enzymes and their substrates are highly specific to each other (enzyme-substrate specificity).

Section 3 of 26Activation energy

Enzymes speed up reactions because they influence the stability of bonds in the reactants. Binding destabilises bonds in the substrate, making it more reactive. Enzymes lower the activation energy of a reaction by providing an alternative energy pathway, so more substrate molecules have enough energy to react at a given temperature.

Section 4 of 26Induced-fit model

In the induced-fit hypothesis the enzyme and its active site (and sometimes the substrate) change shape slightly as the substrate enters, giving a closer fit and stressing the substrate's bonds.

Induced-fit model
This replaced the earlier lock-and-key model in which the active site was a rigid fit.
Scientists use models to explain observations; as technology and research advance, new models develop and old ones can be disproved.

Section 5 of 26Required practical: measuring enzyme activity

The progress of an enzyme-catalysed reaction can be followed by measuring the rate of formation of a product (for example the volume of oxygen released by catalase from hydrogen peroxide, collected in a gas syringe or by displacing water), or the rate of disappearance of the substrate. Keep other variables constant and take repeats.

Section 6 of 26Enzymes and metabolism

Enzymes control metabolism by catalysing particular reactions in sequences called metabolic pathways.

1
Some enzymes work inside cells (intracellular), such as catalase breaking down toxic hydrogen peroxide into water and oxygen,
  • while others work outside cells (extracellular), such as digestive amylase.
2
Because enzymes are not used up they are effective at low concentration.
3
Reactions that would otherwise occur too slowly at body temperature happen rapidly with the right enzyme,
  • allowing organisms to function at temperatures that do not damage their cells.
4
The catalase practical: add hydrogen peroxide to a measured mass of liver or potato, collect the oxygen in a gas syringe and record the volume at regular intervals; the rate is volume divided by time.

Section 7 of 26Worked examples

  1. Explain why an enzyme only catalyses one reaction. [3]3 marks

  2. Explain how an enzyme lowers activation energy. [2]2 marks

  3. Describe the induced-fit model. [2]2 marks

Section 8 of 26Amylase and iodine

In this investigation the rate of substrate disappearance is used to compare rates of reaction under different conditions.

Amylase and iodine
Amylase is a digestive enzyme that hydrolyses starch (the substrate) into maltose and glucose; it functions best at about pH 7 and 37 °C.
At timed intervals drop samples into iodine in potassium iodide on a spotting tile: blue-black means starch remains, orange-brown means it has been digested.
The time taken to reach no colour change is the end point; rate is 1/time.

Section 9 of 26Using a colorimeter

A colorimeter measures light absorbance (how much light is absorbed) or transmission (how much passes through).

Using a colorimeter
Colorimetry can be used in any enzyme-catalysed reaction that involves a colour change.
For example, it can follow the starch-amylase reaction as starch breaks down: the blue-black colour with iodine fades,
  • so absorbance falls and transmission rises.
A weak iodine solution is used to calibrate the colorimeter, and the end point is 100% transmission.

Section 10 of 26Variables and design

Enzyme rate experiments determine the effect of changing one particular variable on the reaction rate.

Variables and design
Variables include temperature, pH, enzyme concentration, substrate concentration and inhibitor concentration.
Only one may be changed (independent variable); the rest are controlled, using a water bath for temperature, buffers for pH and measured volumes for concentrations.
Repeats and a control (for example boiled enzyme) improve reliability.

Section 11 of 26Drawing graphs and finding initial rate

Use appropriate linear scales on axes, plot the independent variable on the x-axis with units, plot points accurately and join with a line of best fit or smooth curve.

For linear graphs the gradient is the same throughout, so rate of change = change ÷ time.
Tangent
For a curve, draw a tangent at t = 0 to find the initial rate: gradient = change in y ÷ change in x.
If a tangent shows 72 cm3 of product in the first 20 s, the initial rate is 72 ÷ 20 = 3.6 cm3 s-1.
The initial rate is used because later the substrate concentration has fallen and product may inhibit the reaction.

Section 12 of 26Common sources of error

In the amylase-iodine practical the end point is judged by eye, which is subjective; a colorimeter reduces this.

Common sources of error
Enzyme and substrate must reach the target temperature before mixing, otherwise the reaction begins at the wrong temperature.
Timing errors, carrying over enzyme on the pipette into the iodine and using different volumes of drops all reduce reliability.
Reduce uncertainty by using larger volumes, standardised drop sizes and at least three repeats of each condition, calculating a mean and comparing the spread to spot anomalies.

Section 13 of 26Worked examples

  1. Describe how you would find the time for amylase to digest starch at different temperatures. [5]5 marks

  2. A tangent shows 72 cm3 of product formed in 20 s. Calculate the initial rate. [2]2 marks

  3. Explain why a colorimeter is calibrated with weak iodine solution. [2]2 marks

Section 14 of 26Temperature

Enzymes have an optimum temperature at which they catalyse at the maximum rate.

At lower temperatures reactions are slow or do not proceed: molecules have less kinetic energy,
  • so there is a lower frequency of successful collisions between substrate and active site.
As temperature rises the rate increases because molecules move faster and collide more often with more energy.
Denatured
Above the optimum, vibration breaks the hydrogen and ionic bonds holding the tertiary structure, the active site changes shape and the enzyme is denatured: substrate no longer fits,
  • and the rate drops sharply.
In answers explain how temperature affects the speed of the molecules and so the number of successful collisions.

Section 15 of 26pH

Each enzyme has an optimum pH.

pH
Enzymes are denatured at extremes of pH,
  • because the hydrogen and ionic bonds that hold the tertiary structure are broken by an excess of H+ (acidic) or OH- (alkaline) ions.
Buffer solutions, each with a specific pH that is maintained even if the reaction would otherwise change it, are used to investigate pH.
Temperature affects both molecular movement (collision rate) and denaturation at high levels,
  • whereas pH does not affect collision rate but changes in pH disrupt substrate binding,
    • reducing successful collisions until the active site is no longer functional.

pH = −log10[H+]. Do not forget the minus sign; pH values should be between 0 and 14, and a weak acid should be about pH 4-6. For [H+] = 3.5 × 10-11 mol dm-3, pH = 10.456 (alkaline).

What the symbols mean: pH has no unit · [H+] = concentration of hydrogen ions (mol dm−3); each 1 pH unit is a ten-fold change in [H+]

Section 16 of 26Enzyme concentration

The higher the enzyme concentration, the greater the number of active sites and the greater the likelihood of enzyme-substrate complexes forming. As long as there is enough substrate, the initial rate increases linearly with enzyme concentration. If substrate becomes limiting, the rate levels off.

Section 17 of 26Substrate concentration

The greater the substrate concentration, the higher the rate, because the chance of forming enzyme-substrate complexes increases.

Substrate concentration
With constant enzyme concentration, a point is reached where all active sites are occupied and the enzyme works at its maximum rate, Vmax; extra substrate molecules must wait for active sites to become free,
  • so the graph plateaus.
At that point enzyme concentration is the limiting factor.

Section 18 of 26Worked examples

  1. Explain the shape of the graph of rate against temperature for an enzyme. [5]5 marks

  2. Ethanoic acid from wood ants has [H+] = 8.39 × 10-6 mol dm-3. Find the pH. [2]2 marks

  3. Explain why the rate stops increasing at high substrate concentration. [3]3 marks

Section 19 of 26Reversible inhibitors

An enzyme's activity can be reduced or stopped temporarily by a reversible inhibitor. There are two types. Competitive inhibitors have a shape similar to the substrate and compete with it for the active site, so fewer enzyme-substrate complexes form. Non-competitive inhibitors bind to another site (an allosteric site) on the enzyme, changing the shape of the active site so the substrate can no longer bind.

Section 20 of 26End-product inhibition

Metabolic reactions can be controlled by using the end-product of a sequence of reactions as a non-competitive, reversible inhibitor.

As the enzyme converts substrate to product, the process slows itself because the end-product binds to an alternative site on the original enzyme, changing the shape of the active site and preventing further enzyme-substrate complexes.
The end-product can then detach, allowing the active site to reform and the enzyme to become active again.
End-product inhibition
As product levels fall the enzyme starts catalysing again, forming a continuous feedback loop, called end-product inhibition.

Section 21 of 26Effect on the rate

InhibitorInitial rateVmaxEffect of more substrate
CompetitiveReduced by blocking active sitesStill reached at high substrate concentrationOvercomes the inhibition
Non-competitiveReducedReduced (fewer active enzymes)Does not overcome inhibition

Section 22 of 26Control of variables and uncertainty

In enzyme rate experiments only one factor (temperature, pH, enzyme, substrate or inhibitor concentration) is changed at a time.

Control of variables and uncertainty
If the uncertainty of a measurement is known, the percentage error is (uncertainty ÷ measured value) × 100.
  • For example, with 50 cm3 of oxygen from the breakdown of hydrogen peroxide by catalase and an uncertainty of 0.5 cm3, the percentage error is 0.5 ÷ 50 × 100 = 1%.
Using larger volumes or more precise apparatus reduces percentage error.

Section 23 of 26Examples and exam application

A well-known competitive inhibitor is malonate, which resembles succinate and inhibits succinate dehydrogenase in respiration.

1
Heavy metal ions such as mercury or silver are non-competitive inhibitors that can bind to enzymes at other sites,
  • and many drugs and poisons act as enzyme inhibitors.
2
Statins reduce cholesterol synthesis by inhibiting an enzyme.
3
When drawing graphs: with a competitive inhibitor, the curve of rate against substrate concentration rises more slowly but reaches the same Vmax as the uninhibited enzyme; with a non-competitive inhibitor, the curve plateaus at a lower Vmax.
4
Increasing the inhibitor concentration produces a greater effect in both cases until all enzymes or active sites are blocked.

Section 24 of 26Worked examples

  1. Explain how a competitive inhibitor reduces the rate of reaction. [3]3 marks

  2. Why can high substrate concentration overcome competitive but not non-competitive inhibition? [3]3 marks

  3. Calculate the percentage error for 50 cm3 of oxygen with an uncertainty of 0.5 cm3. [2]2 marks

  4. Describe end-product inhibition and why it is useful. [3]3 marks

Section 25 of 26Key terms

TermMeaning
EnzymeBiological catalyst, usually a protein
Active siteRegion of an enzyme where the substrate binds
Enzyme-substrate complexTemporary structure formed when substrate binds
Activation energyEnergy needed to start a reaction
Induced-fitActive site adapts its shape to the substrate
TermMeaning
AmylaseEnzyme that hydrolyses starch to maltose
ColorimeterInstrument measuring light absorbed or transmitted
Initial rateRate at the very start, found from a tangent
Independent variableVariable deliberately changed
Control variableVariable kept constant
TermMeaning
OptimumCondition giving the maximum rate of reaction
DenaturationIrreversible change of tertiary structure and active site shape
BufferSolution that maintains a constant pH
VmaxMaximum rate when all active sites are saturated
Limiting factorFactor in shortest supply that restricts the rate
TermMeaning
Competitive inhibitorBinds to the active site, competing with substrate
Non-competitive inhibitorBinds elsewhere, altering the active site
End-product inhibitionProduct inhibits an earlier enzyme in its pathway
Percentage error(Uncertainty ÷ measured value) × 100
Feedback loopOutput of a process regulates the process

Section 26 of 26Exam technique

1
Saying enzymes are used up in the reaction.
2
Describing the substrate as changing the active site shape permanently.
3
Saying enzymes provide energy for reactions.
4
Using 'lock and key' as if the active site were rigid for all enzymes.
1
Changing more than one variable in a rate experiment.
2
Drawing the tangent at the end of the reaction.
3
Not using buffers when investigating temperature.
4
Mixing up absorbance and transmission when starch is digested.
1
Saying enzymes are killed by high temperature; they are denatured.
2
Saying low temperature denatures enzymes; it only slows them.
3
Forgetting to mention that enzyme-substrate complexes form less often.
4
Omitting the minus sign in pH = −log[H+].
1
Saying non-competitive inhibitors bind to the active site.
2
Believing inhibitors always denature the enzyme.
3
Forgetting that competitive inhibition can be overcome by more substrate.
4
Dividing uncertainty by the wrong value in percentage error.

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