IB DP Biology Topic 2: Molecular biology 2.5 Enzymes Study Notes

2.5  Enzymes

Essential Idea:
Enzymes control the metabolism of the cell     

Understandings:

  • Enzymes have an active site to which specific substrates bind
  • Enzyme catalysis involves molecular motion and the collision of substrates with the active site
  • Temperature, pH and substrate concentration affect the rate of activity of enzymes
  • Enzymes can be denatured
  • Immobilized enzymes are widely used in industry

Applications:

  • Methods of production of lactose-free milk and its advantages

Skills:

  • Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes
  • Experimental investigation of a factor affecting enzyme activity

2.5.U1  Enzymes have an active site to which specific substrates bind.

  • State the relationship between enzyme substrate and enzyme active site.
  • Explain the relationship between enzyme structure and enzyme specificity, including the role of the active site.​

2.5.U2  Enzyme catalysis involves molecular motion and the collision of substrates with the active site.

  • Outline the three stages of enzyme activity.
  • Explain the role of random collisions in the binding of the substrate with the enzyme active site.
  • Describe the induced fit model of enzyme action.​

2.5.U3  Temperature, pH and substrate concentration affect the rate of activity of enzymes.

  • Explain how temperature affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of temperature on the rate of enzyme activity.
  • Explain how pH affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of pH on the rate of enzyme activity.
  • Identify the optimum temperature or pH for enzyme activity on a graph.
  • Explain how substrate concentration affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of substrate concentration on the rate of enzyme activity.​

2.5.U4  Enzymes are denatured.

  • State the effect of denaturation on enzyme structure and function.

2.5.U5  Immobilized enzymes are widely used in industry.

  • List industries that use commercially useful enzymes.
  • Explain how and why industrial enzymes are often immobilized.

2.5.A1  Methods of production of lactose-free milk and its advantages.

  • State the source of the lactase enzyme used in food processing.
  • State the reaction catalyzed by lactase.
  • Outline four reasons for using lactase in food processing.​

2.5.S1  Design of experiments to test the effect of temperature, pH, and substrate concentration on the activity of enzymes.

  • Identify and manipulated, responding and controlled variables in descriptions of experiments testing the activity of enzymes.

2.5.S2  Experimental investigation of a factor affecting enzyme activity. (Practical 3)

  • Describe three techniques for measuring the activity of an example enzyme.

2.5.NOS  Experimental design-accurate, quantitative measurements in enzyme experiments require replicates to ensure reliability.

  • Define quantitative and qualitative.
  • Determine measurement uncertainty of a measurement tool.
  • Explain the need for repeated measurements (multiple trials) in experimental design.
  • Explain the need to controlled variables in experimental design.

Topic 2.5 enzymes

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In the Enzyme unit students learn about the structure of the enzyme and the factors affecting enzyme activity.

​The unit is planned to take 2  school days


​Essential idea:

  • Enzymes control the metabolism of the cell.

Nature of science:

  • Experimental design—accurate, quantitative measurements in enzyme experiments require replicates to ensure reliability. (3.2)
    • Define quantitative and qualitative.
    • Determine measurement uncertainty of a measurement tool.
    • Explain the need for repeated measurements (multiple trials) in experimental design.
    • Explain the need to controlled variables in experimental design
Understanding:

2.5.U1 Enzymes have an active site to which specific substrates bind.

  • State the relationship between enzyme substrate and enzyme product.
  • Explain the relationship between enzyme structure and enzyme specificity, including the role of the active site.​

An enzyme is a globular protein which acts as a biological catalyst by speeding up the rate of a chemical reaction. Enzymes are not changed or consumed by the reactions they catalyse and thus can be reused. Enzymes are typically named after the molecules they react with (called the substrate) and end with the suffix ‘-ase’. The active site is the region on the surface of the enzyme which binds to the substrate molecule. The active site and the substrate complement each other in terms of both shape and chemical properties. Only a specific substrate is capable of binding to a particular enzyme’s active site

  • work as globular proteins that work as catalysts = speed up chemical reactions without being altered themselves
  • substances that enzymes convert into products in these reactions is called substrates – general equation for an enzyme-catalysed reaction is substrate (enzyme–>) product
  • many different enzymes are needed because they only catalyse one biochemical reaction and thousands of reactions take place in cells nearly all of which need to be catalysed – enzyme-substrate specificity
  • look at the mechanism by which enzymes speed up reactions – substrate, substrates binding to a special region on the surface of the enzyme called the active site
  • shape and chemical properties of the active site and the substrate match each other = allows the substrate to bind not other substances
  • substances are converted into products while they are bound to the active site and the products are then released, freeing the active site to catalyse another reaction

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2.5.U2 Enzyme catalysis involves molecular motion and the collision of substrates
with the active site.
  • Outline the three stages of enzyme activity.
  • Explain the role of random collisions in the binding of the substrate with the enzyme active site.
  • Describe the induced fit model of enzyme action.​

Enzyme reactions typically occur in aqueous solutions. Consequently, the substrate and enzyme are usually moving randomly within the solution. This is referred to as Brownian motion. Sometimes an enzyme may be fixed in position (e.g. membrane-bound) – this serves to localise reactions to particular sites

Stages of enzyme activity

  1. substrate binds to the active site of the enzyme – some enzymes have two substrates that bind to different parts of the active site
  2. while the substrates are bound to the active site they change into different chemical substances, which are the product of the reaction
  3. the products separate from the active site, leaving it vacant for substrates to bind again
  4. the substrate molecules can only bind to the active site if it moves very close to it – coming together of a substrate molecules and an active site is known as collision
  5. most reactions the substrates are dissolved in water around the enzyme because water is in a liquid states its molecules and all the particles dissolved in it are in contact with each other and are in continual motion – each particle can move separately
  6. the direction fo movement changes and is random, basis of diffusion in liquids
  7. both substrates and enzymes with active sites are able to move, though most substrate molecules are smaller than the enzyme so their movement is faster
  8. collisions between substrate molecules and the active site occur because of random movements of both substrate and enzyme – the substrate may be at any angle to the active site when the collision occurs. successful collisions are ones in which the substrate and active site are correctly aligned to allow binding to take place
 

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​2.5.U3 Temperature, pH and substrate concentration affect the rate of activity of
enzymes
.
  • Explain how temperature affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of temperature on the rate of enzyme activity.
  • Explain how pH affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of pH on the rate of enzyme activity.
  • Identify the optimum temperature or pH for enzyme activity on a graph.
  • Explain how substrate concentration affects the rate of enzyme activity.
  • Draw a graph of depicting the effect of substrate concentration on the rate of enzyme activity.​

The activity of an Enzyme is affected by its environmental conditions. Changing these alter the rate of reaction caused by the enzyme. In nature, organisms adjust the conditions of their enzymes to produce an Optimum rate of reaction, where necessary, or they may have enzymes which are adapted to function well in extreme conditions where they live.

Temperature

  • Low temperatures result in insufficient thermal energy for the activation of an enzyme-catalysed reaction to proceed
  • Increasing the temperature will increase the speed and motion of both enzyme and substrate, resulting in higher enzyme activity
  • This is because a higher kinetic energy will result in more frequent collisions between the enzymes and substrates
  • At an optimal temperature (may vary for different enzymes), the rate of enzyme activity will be at its peak
  • Higher temperatures will cause enzyme stability to decrease, as the thermal energy disrupts the enzyme’s hydrogen bonds
  • This causes the enzyme (particularly the active site) to lose its shape, resulting in the loss of activity (denaturation)

The temperature at which the maximum rate of reaction occurs is called the enzyme’s Optimum Temperature. This is different for different enzymes. Most enzymes in the human body have an Optimum Temperature of around 37.0 °C.

pH – Acidity and Basicity
pH measures the Acidity and Basicity of a solution. It is a measure of the Hydrogen Ion (H+) concentration, and therefore a good indicator of the Hydroxide Ion (OH-) concentration. It ranges from pH1 to pH14. Lower pH values mean higher H+ concentrations and lower OH- concentrations.

  • Changing the pH will alter the charge of the enzyme, which in turn will alter protein solubility and overall shape
  • Changing the shape or charge of the active site will diminish its ability to bind the substrate, abrogating enzyme function
  • Enzymes have an optimal pH (may differ between enzymes) and moving outside this range diminishes enzyme activity
  • Small changes in pH above or below the Optimum do not cause a permanent change to the enzyme, since the bonds can be reformed. However, extreme changes in pH can cause enzymes to Denature and permanently lose their function.

Enzymes in different locations have different Optimum pH values since their environmental conditions may be different. For example, the enzyme Pepsin functions best at around pH2 and is found in the stomach, which contains Hydrochloric Acid (pH2).

Concentration

  • Changing the Enzyme and Substrate concentrations affect the rate of reaction of an enzyme-catalysed reaction. Controlling these factors in a cell is one way that an organism regulates its enzyme activity and so its Metabolism.
  • Changing the concentration of a substance only affects the rate of reaction if it is the limiting factor: that is, it the factor that is stopping a reaction from preceding at a higher rate.

If it is the limiting factor, increasing concentration will increase the rate of reaction up to a point, after which any increase will not affect the rate of reaction. This is because it will no longer be the limiting factor and another factor will be limiting the maximum rate of reaction. As a reaction proceeds the rate of reaction will decrease, since the Substrate will get used up. The highest rate of reaction, known as the Initial Reaction Rate is the maximum reaction rate for an enzyme in an experimental situation.

Substrate Concentration
Increasing Substrate Concentration increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed.

  • Increasing substrate concentration will increase the activity of a corresponding enzyme
  • More substrates mean there is an increased chance of enzyme and substrate colliding and reacting within a given period
  • After a certain point, the rate of activity will cease to rise regardless of any further increases in substrate levels
  • This is because the environment is saturated with substrate and all enzymes are bound and reacting (Vmax)

Enzyme Concentration

  • Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will be colliding with substrate molecules.
  • However, this too will only have an effect up to a certain concentration, where the Enzyme Concentration is no longer the limiting factor..

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​2.5.U4 Enzymes can be denatured.

  • State the effect of denaturation on enzyme structure and function.​
The important part of an enzyme is called the active site. This is where specific molecules bind to the enzyme and the reaction occurs.
Anything that changes the shape of the active site stops the enzyme from working. This is similar to a key that opens a door lock. It does not matter what a key handle looks like, but if you change the shape of the ‘teeth’ the key no longer works.

The shape of the active site is affected by pH. This is why enzymes will only work at a specific pH, as well as a specific temperature. Change the pH and the enzyme stops working.
Increasing the temperature to 60°C will cause a permanent change to the shape of the active site. This is why enzymes stop working when they are heated. We say they have become denatured.
 

Protein Denature Animation

 

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​2.5.U5 Immobilized enzymes are widely used in industry.

  • List industries that use commercially useful enzymes.
  • Explain how and why industrial enzymes are often immobilized

Enzyme immobilization is the process of confining the enzyme molecule to a distinct phase from the one where in the substrates and the products are present. This allows the enzyme to retain its catalytic activity and be repeatedly and continuously used.

Applications of enzyme immobilization

  • Industrial production – industrial production of antibiotics, beverages, amino acids etc. uses immobile enzymes or who cells
  • Biomedical applications – immobilized enzymes are widely used in the diagnosis and treatment of many diseases. Immobilized enzymes can be used to overcome inborn metabolic disorders by the supply of immobilized enzymes. Mobilization techniques are effectively used in drug delivery systems especially to oncogenic sites
  • Food industry – enzymes like pectinases and cellulases immobilized on suitable carriers are successfully used in the production of jams, jellies and syrups from fruits and vegetables
  • Research – a research activity extensively uses many enzymes. The use of immobilized enzyme allows researchers to increase the efficiency of different enzymes such as Horse Radish Peroxidase (HRP) in blotting experiments and different Proteases for cell or organelle lysis
  • Production of biodiesel from vegetable oils
  • Wastewater management – treatment of sewage and industrial effluents.
  • Textile industry – scouring, bio-[polishing and desizing of fabrics
  • Detergent industry – immobilization of lipase enzyme for effective dirt removal from clothes

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Application & Skill:

2.5.A1 Methods of production of lactose-free milk and its advantages.

  • State the source of the lactase enzyme used in food processing.
  • State the reaction catalyzed by lactase.
  • Outline four reasons for using lactase in food processing.​

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Lactose is the sugar found in milk. It can be broken down by the enzyme lactase into glucose and galactose. However some people lack this enzyme and so cannot break down lactose leading to lactose intolerance. Lactose intolerant people need to drink milk that has been lactose reduced. Lactose-free milk can be made in two ways. The first involves adding the enzyme lactase to the milk so that the milk contains the enzyme. The second way involves immobilizing the enzyme on a surface or in beads of a porous material. The milk is then allowed to flow past the beads or surface with the immobilized lactase. This method avoids having lactase in the milk.

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2.5.S1 Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. (from BioNinja)

  • Identify and manipulated, responding and controlled variables in descriptions of experiments testing the activity of enzymes

When designing an experiment to test the effect of factors affecting enzyme activity, the three key decisions to be made are:

  • Which factor to investigate (i.e. the independent variable)
  • Which enzyme / substrate reaction to use
  • How to measure the enzyme activity (i.e. the dependent variable)

Choosing the Independent Variable

The main factors which will affect the activity of an enzyme on a given substrate are:

  • Temperature (use water baths to minimise fluctuations)
  • pH (acidic or alkaline solutions)
  • Substrate concentration (choose range to avoid saturation)
  • Presence of inhibitor (type of inhibitor will be enzyme-specific)

Selecting an Enzyme and Substrate

  • Selection will depend on availability within the school, however certain enzymes can be extracted from common food sources

Examples of common enzyme-catalysed reactions include:

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Measuring Enzyme Activity

The method of data collection will depend on the reaction occurring – typically most reactions are measured according to:

  • The amount / rate of substrate decomposition (e.g. breakdown of starch)
  • The amount / rate of product formation (e.g. formation of maltose)

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2.5.S2 Experimental investigation of a factor affecting enzyme activity.
(Practical 3)
  • Describe three techniques for measuring the activity of an example enzyme.
Key things to consider when conducting an experimental investigation into a factor affecting enzyme activity include:
  • What is an appropriate range of values to select for your independent variable?
  • Have you chosen a sufficient time period for the reaction to proceed?
  • Have you identified, and controlled, all relevant extraneous variables?
  • Can you include a negative control condition (no enzyme) to establish baseline readings?
  • Can you include a positive control condition to confirm enzyme activity?
  • Is it possible to treat the enzyme with the independent variable before mixing with the substrate?
  • Does the data collection method allow for sufficient precision in detecting changes to levels of product / substrate?
  • Have all appropriate safety precautions been taken when handling relevant substances?

Solved Examples: Enzymes

3.6.1 Define enzyme and active site.

Enzymes: Globular proteins which act as catalysts of chemical reactions. 
Active site: Region on the surface of an enzyme to which substrates bind and which catalyses a chemical reaction involving the substrates.

3.6.2 Explain enzyme?substrate specificity.

The active site of an enzyme is very specific to its substrates as it has a very precise shape. This results in enzymes being able to catalyze only certain reactions as only a small number of substrates fit in the active site. This is called enzyme-substrate specificity. For a substrate to bind to the active site of an enzyme it must fit in the active site and be chemically attracted to it. This makes the enzyme very specific to it’s substrate. The enzyme-substrate complex can be compared to a lock and key, where the enzyme is the lock and the substrate is the key.

3.6.3 Explain the effects of temperature, pH and substrate concentration on enzyme activity.

Enzyme activity increases with an increase in temperature and usually doubles with every 10 degrees rise. This is due to the molecules moving faster and colliding more often together. However at a certain point the temperature gets to high and the enzymes denature and stop functioning. This is due to the heat causing vibrations within the enzyme destroying its structure by breaking the bonds in the enzyme.

Enzymes usually have an optimum pH at which they work most efficiently. As the pH diverges from the optimum, enzyme activity decreases. Both acid and alkali environments can denature enzymes.

Enzyme activity increases with an increase in substrate concentration as there are more random collisions between the substrate and the active site. However, at some point, all the active sites are taken up and so increasing the substrate concentration will have no more effect on enzyme activity. As long as there are active sites available, an increase in substrate concentration will lead to an increase in enzyme activity.

3.6.4 Define denaturation.

Denaturation is changing the structure of an enzyme (or other protein) so it can no longer carry out its function.

3.6.5 Explain the use of lactase in the production of lactose-free milk.

Lactose is the sugar found in milk. It can be broken down by the enzyme lactase into glucose and galactose. However some people lack this enzyme and so cannot break down lactose leading to lactose intolerance. Lactose intolerant people need to drink milk that has been lactose reduced. Lactose-free milk can be made in two ways. The first involves adding the enzyme lactase to the milk so that the milk contains the enzyme. The second way involves immobilizing the enzyme on a surface or in beads of a porous material. The milk is then allowed to flow past the beads or surface with the immobilized lactase. This method avoids having lactase in the milk.

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