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1: Biochemistry of macromolecules and metabolic pathways

Enzymes are referred to as biological catalysts – they create new pathways that allow reactions to occur many times faster than uncatalysed reactions.

Enzymes act on specific molecules called substrates. These substrate molecules bind to a region of the enzyme known as the active site. The specificity of the enzyme is due to the fact that the substrate and active site have structures that are complementary. Hence only specific substrates can fit and bind to the active site.

On successful completion of this topic you will: • understand the chemical principles that apply to the structures of

biological building block molecules (LO1) • understand the structures of biological macromolecules and the

relationships to biological functions (LO2).

To achieve a Pass in this unit you need to show that you can: • explain the structure, catalytic function and characteristic properties of

enzymes (2.2).

Enzymes1.2

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1: Biochemistry of macromolecules and metabolic pathways

1.2: Enzymes

1 Models to explain the actions of enzymesBefore you startIf you find some parts of this unit challenging, remember you are working at a higher level than you may be used to. In this unit it is important that you fully understand the following themes and topics before you begin:

• structure and function of biological molecules • enzyme structure and function • aerobic respiration.

If you need to check your understanding of proteins, carbohydrates, lipids and nucleic acids, Unit 2 Module 1 of OCR AS Biology (P. Kennedy and F. Sochacki, 2008), offers a good introduction to the topic.

If you need to check your understanding of aerobic respiration and the stages of glycolysis, link reaction, the Krebs cycle and the electron transport chain, you may find Unit 1 Module 4 of OCR A2 Biology (S. Hocking, 2008) useful.

Several scientists have produced models to help explain the actions of enzymes.

The lock and key hypothesisThis model explains that enzyme activity depends on the active site of the enzyme. The active site is the area or ‘pocket’ on the enzyme where a substrate molecule fits. The shapes of the substrate molecule and the active site are complementary, so they are said to fit together like a key fits into a lock. When the substrate binds to the active site an enzyme-substrate complex is formed and the substrate then forms a product.

The induced-fit hypothesisThis model, in contrast to the lock and key hypothesis, suggests that the active site is not exactly the same shape as the substrate – it is said to be in a ‘relaxed’ state. When the substrate binds to the active site, the active site moulds itself to the substrate forming an enzyme-substrate complex. Only then is the active site the correct shape to catalyse the reaction.

Substrate

Active site

Schematic modelof an enzyme

Molecular modelof catalase

Substrate = H2O2

Active site

The schematic diagram in Figure 1.2.1 shows the complimentary active site of an enzyme and the substrate. The molecular diagram shows the enzyme catalase and its complementary substrate hydrogen peroxide.

Key termsEnzyme: A protein used as a catalyst to speed up chemical reactions.

Active site: The functional part of the enzyme.

Substrate: A substance that binds to an enzyme’s active site; it is the reactant molecule.

Figure 1.2.1: Enzyme structure.

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1: Biochemistry of macromolecules and metabolic pathways

1.2: Enzymes

2 Lowering activation energyFor chemical reactions to take place bonds need to be broken before new ones can be formed. The energy needed for these bonds to be broken is known as the activation energy and this is usually very high. An enzyme carries out its function by providing an alternative route for the reaction with a lower activation energy by temporarily combining with the chemicals involved in the reaction. Figure 1.2.2 is an energy profile showing the effect of a catalyst on activation energy.

Ener

gy

Progress of reaction

Activation energywithout catalyst

Activation energywith catalyst

Reactants

Products

3 Factors affecting enzyme activityThere are various factors that affect enzyme activity and all enzymes have their own optimum conditions. Enzyme rates depend on the surrounding conditions and substrate concentration. Conditions that affect enzyme activity therefore are temperature, pH, substrate and enzyme concentration and the presence of any inhibitors.

TemperatureApplying heat energy to molecules increases their kinetic energy so there will be an increased number of collisions between enzyme and substrate molecules. This in turn will increase the rate of reaction and so the products will be formed more quickly. However, applying too much heat can cause enzymes to denature. The increased vibrations and collisions put strains on the bonds of the tertiary structure and can break the hydrogen and ionic bonds. The breaking of these bonds affects the important three-dimensional shape; more importantly it changes the shape of the active site. Denaturation causes the enzyme to lose its ability to function and its function cannot be restored.

Increasing the temperature initially increases the rate of reaction. The temperature that gives the maximum rate of reaction is the enzyme’s optimum temperature. However, the rate will decrease if the temperature rises higher than the optimum, due to denaturation of the enzyme. Figure 1.2.3 shows the effect of temperature on the rate of an enzyme-controlled reaction.

Figure 1.2.2: The activation energy needed for a chemical reaction to take

place is lower when a catalyst is present.

Key termsActivation energy: Energy required to activate a chemical reaction.

Denature: To change an enzyme’s structure in a way that changes the shape of the active site so the enzyme is unable to function.

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1: Biochemistry of macromolecules and metabolic pathways

1.2: Enzymes

Enzymes inside the body work at an optimum temperature of 37 °C, whereas genetic engineering techniques require high temperatures, and the enzyme DNA polymerase has an optimum temperature of 72 °C. Under optimum conditions and at this temperature DNA polymerase is able to efficiently polymerise a thousand bases per minute. The amount of the target DNA sequence is doubled, leading to exponential amplification of the specific DNA fragment.

0 10 20 30 40 50 60 70

Temperature/°C

Rate

of r

eact

ion

Opt

imum

tem

pera

ture

giv

esm

axim

um ra

te o

f rea

ctio

n

Increasing temperatureincreases the rate ofreaction due to increasedkinetic energy

Increasing temperaturebeyond the optimumtemperature reducesthe rate of reaction dueto the breaking of bondsholding the enzyme’stertiary structure in place

pHThe optimum pH varies for all enzymes – for many enzymes the optimum pH is pH 7. However, outside their optimum pH range, enzymes are denatured because free hydrogen and hydroxide ions affect the charge of the amino acid. The three-dimensional shape is altered, changing the tertiary structure of the protein and, in turn, the specific active site.

Pepsin is an enzyme that works in the human body to digest proteins; it works in the stomach at pH 2. Trypsin, an enzyme in the small intestine, also digests proteins – however, in contrast, this enzyme works at pH 7.

Substrate concentration When you look at Figure 1.2.4 you can see that as the substrate concentration increases, the rate of the enzyme-controlled reaction increases until all the enzymes’ active sites are occupied by substrate. We use the term ‘V

max’, which is

the maximum rate at which an enzyme catalyses a reaction and is often known as the turnover rate. When all the enzymes’ active sites are saturated with a complementary substrate V

max is reached. The only way to speed up the reaction

further is to add more enzyme. The amount of substrate needed to achieve Vmax

is important and is often expressed as Michaelis-Menten constant, K

m, which is

the substrate concentration needed for an enzyme to reach one half its maximum rate. All enzymes have their own specific K

m for a substrate.

Figure 1.2.3: Effect of temperature on the rate of an enzyme-controlled reaction.

Key termsTurnover rate: The number of substrate molecules converted into product molecules per unit time, known as V

max.

Michaelis-Menten constant: The substrate concentration needed for an enzyme to reach one half of its maximum rate known as K

m. The

catalysed reaction rate is equal to V

max /2.

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1: Biochemistry of macromolecules and metabolic pathways

1.2: Enzymes

Reac

tion

vel

ocit

y (V

0)

Substrate concentration (S)

Vmax

Vmax/2

Km

Enzyme concentrationEnzymes can convert millions of substrate molecules into products every minute, so the number of enzymes required in a reaction is less than the substrate. However, if there is a plentiful supply of substrate, the reaction can be limited by the number of enzymes present; therefore, by increasing the enzyme concentration, the rate will also increase.

Remember that if there is not an abundant supply of substrate, increasing the concentration of enzyme will have no effect on the rate of reaction.

ActivityAnswer the following questions:1 How does temperature affect enzyme activity?2 How does pH affect enzyme activity?3 If all the enzymes’ active sites are occupied, what effect would adding more substrate have on

the rate of reaction and why?4 If there was a plentiful supply of substrate how could the rate of reaction be increased?5 What does denature mean and how does it occur?

Presence of inhibitorsEnzymes: competitive inhibitors

As the name suggests, competitive inhibitors compete with the substrate for the active site of the enzyme. Therefore the inhibitors are the same size and shape as the substrate to enable the complementary fit inside the enzyme’s active site. However, the inhibitor cannot form a product. If competitive inhibitors are present, the rate of reaction is decreased as they stop the substrate from binding to the enzyme and producing a product. Many competitive inhibitors do not bind permanently and so their action is reversible. They bind to the enzyme’s active site for a short period and then detach, leaving the enzyme active again.

Enzymes: non-competitive inhibitors

These inhibitors do not compete for the active site but they bind to an alternative part of the enzyme called an allosteric region. This changes the shape of the active site and means that the substrate will no longer be a complementary shape to fit into the active site. If there are enough inhibitors present they may fill all the enzymes’ allosteric regions and, if this happens, the reaction will stop.

Figure 1.2.4: A graph to show V

max and K

m.

LinkYou will find out more about enzymes in Topic guide 1.4: Investigating enzymes. Also check out Unit 5: Chemistry for applied biologists, for enzyme kinetics.

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1: Biochemistry of macromolecules and metabolic pathways

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Figure 1.2.5 shows competitive inhibition and non-competitive inhibition taking place.

Competitive inhibitorinterferes with activesite of enzyme sosubstrate cannot bind

(a) Competitive inhibition (b) Non-competitive inhibition

Non-competitive inhibitor changesshape of enzyme so it cannot bindto substrate

Substrate

Substrate

Enzyme

Enzyme

Permanent inhibitors

The effect of many non-competitive inhibitors is not reversible because they bind permanently to the enzymes; the enzyme is therefore described as denatured because the shape of the active site is no longer complementary or the correct shape for its specific substrate to bind to.

ChecklistIn this topic you should now be familiar with the following ideas about enzymes:

enzymes are biological catalysts

enzymes are proteins

enzymes have an active site that enables them to bind to substrate molecules

two models can be used to explain enzyme action – the lock and key hypothesis and the induced-fit model

enzymes can be affected by temperature and pH

an enzyme’s shape can be altered, which stops the enzyme working; this is known as denaturation

the rate of reaction can be affected by the concentration of the substrate and the enzyme

Vmax

is the maximum rate reached in an enzyme-controlled reaction

competitive inhibitors affect the rate of reaction by competing with the substrate molecules

non-competitive inhibitors affect the rate of reaction by binding to an alternative region and changing the active site shape.

The following Case study is a real life example of enzymes in action.

Case studyIn biochemistry, fermentation is used to produce alcohol, yoghurt, vinegar and many other everyday products. Alcoholic and glycolysis fermentation both begin with glucose and are both anaerobic fermentation processes (do not require oxygen). Glycolysis uses many enzymes to transform glucose to lactic acid. Alcoholic fermentation follows the same enzymatic pathway; however, lactate dehydrogenase is replaced by pyruvate decarboxylase and alcoholic dehydrogenase. During alcoholic fermentation these two enzymes convert pyruvic acid into carbon dioxide and ethanol.

Figure 1.2.5: Competitive inhibition and non-competitive inhibition.

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1: Biochemistry of macromolecules and metabolic pathways

1.2: Enzymes

Further readingBoyle, M. & Senior, K. (2008) Biology, 3rd Edition, HarperCollins

Kennedy, P., Sochacki, F. & Hocking, S. (2008) OCR Biology AS, Heinemann (Pearson Education Limited)

Kennedy, P., Sochacki, F., Winterbottom, M. & Hocking, S. (2008) OCR Biology A2, Heinemann (Pearson Education Limited)

Loomis, H.F. (2005) Enzymes: The Key to Health, 21st Century Nutrition Publishing

Moran, L., Horton, R., Scrimgeour, G., Perry, M. & Rawn, D. (2011) Principles of Biochemistry (International Edition), 5th Edition, Pearson

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Figure 1.2.3: Table showing the effect of temperature on the rate of an enzyme-controlled reaction from Figure 4 on page 129 of Pearson’s AS Biology for OCR. Used with permission.

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