Topic 2.5: Enzymes

topic 2.5 enzymes

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

Enzyme Animation

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

substrate binds to the active site of the enzyme – some enzymes have two substrates that bind to different parts of the active site
while the substrates are bound to the active site they change into different chemical substances, which are the product of the reaction
the products separate from the active site, leaving it vacant for substrates to bind again
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
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
the direction fo movement changes and is random, basis of diffusion in liquids
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
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

Biochemical Pathway Animation

Feedback Inhibition Animation

image from BBC Bitesize

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..

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

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

image from RSC Publishing - Royal Society of Chemistry

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.

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.

image fromBourgeois Essence

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:

image from BioNinja

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)

image from BioNinja

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?

Go to Internatal Assessment

Key Term:

enzyme
globular shape
catalyst

active site
amino acid
denature

substrate
covalent bond
immobilized

denaturation
lactose

lactase
enzyme specificity

Class Material:

Enzyme Technique Lab
Factors Affecting Enzymes (student designed)

Topic 2.5 Review

Powerpoint and Notes on Topic 2.5 by Chris Payne

Correct use of terminology is a key skill in Biology. It is essential to use key terms correctly when communicating your understanding, particularly in assessments. Use the quizlet flashcards or other tools such as learn, scatter, space race, speller and test to help you master the vocabulary.

Useful Links:

KScience
Lew Ports Biology Page
Chemistry for Kids
Enzyme Lab
Clear and simple animations, with factors from KScience.co.uk
More simple animations from John Gianni
How enzymes work from McGraw Hill
What is an enzyme? from Northland (the best one – including inhibitors, pathways and feedback inhibition)
A full collection of savable enzyme animations from Husam Medical
Enzyme action and the hydrolysis of sucrose from McGraw Hill
Protein denaturation from McGraw Hill
The pH scale PhET Lab (allow Java to run)

News:
Cancer Cell Enzymes BBC News Health
New Small Molecule Catalyst Phys.org

Video Clips:

Paul Andersen explains how enzymes are used to break down substrates. The correct shape of the active site allows a key/lock fit between the enzyme and the substrate. The enzyme catalase is used to break down hydrogen peroxide. The importance of cofactors and coenzymes is emphasized. Competitive and allosteric inhibition is also included.

Dr. Kiki breaks down the breakdown of proteins.

All adult mammals but humans are lactose intolerant. Follow human geneticist Spencer Wells, director of the Genographic Project of the National Geographic Society, as he tracks down the genetic and societal changes associated with the ability to digest lactose as adults—or lactose tolerance

Reference sites

i-Biology
Click4Biology
Online IB Biology Subject Guide
BioNinja
Biology For Life
IB Biology Help