Enzymes control the metabolism of the cell. Topic 2.5 IB Biology Miss Werba

TOPIC 2 – MOLECULAR BIOLOGY 2.1 MOLECULES TO METABOLISM 2.2 WATER 2.3 CARBOHYDRATES & LIPIDS 2.4 PROTEINS 2.5 ENZYMES 2.6 STRUCTURE OF DNA & RNA 2.7 DNA REPLICATION, TRANSCRIPTION & TRANSLATION 2.8 CELL RESPIRATION 2.9 PHOTOSYNTHESIS J WERBA – IB BIOLOGY 2

THINGS TO COVER Statement Guidance U.1 U.2 U.3 U.4 U.5 A.1 S.1 S.2 Enzymes have an active site to which specific substrates bind. U.2 Enzyme catalysis involves molecular motion and the collision of substrates with the active site. U.3 Temperature, pH and substrate concentration affect the rate of activity of enzymes. Students should be able to sketch graphs to show the expected effects of temperature, pH and substrate concentration on the activity of enzymes. They should be able to explain the patterns or trends apparent in these graphs. U.4 Enzymes can be denatured. U.5 Immobilized enzymes are widely used in industry. A.1 Methods of production of lactose-free milk and its advantages. Lactase can be immobilized in alginate beads and experiments can then be carried out in which the lactose in milk is hydrolysed. S.1 Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. S.2 Experimental investigation of a factor affecting enzyme activity (Practical 3). NOS 3.2 Experimental design J WERBA – IB BIOLOGY 3

ENZYMES Biological catalysts, which speed up the rate of chemical reactions without becoming a part of the products. J WERBA – IB BIOLOGY 4

ENZYMES Enzymes are globular proteins with a unique shape. Without enzymes, chemical reactions would occur very slowly. Enzymes work by lowering the activation energy needed for a reaction. Many enzyme names end in –ase eg. amylase, lipase, sucrase, lactase, catalase J WERBA – IB BIOLOGY 5

ACTIVE SITES U.1 The region on an enzyme’s surface to which a substrate binds. J WERBA – IB BIOLOGY 6

ACTIVE SITES The active sites are specific: U.1 The active sites are specific: ie. enzymes are designed to “fit” the shape of their substrate This is known as the Lock and Key Model The active site is simply a groove or cleft on the surface of the enzyme J WERBA – IB BIOLOGY 7

ENZYME-SUBSTRATE SPECIFICITY Active site and substrate complement each other in terms of both shape and chemical properties (e.g. opposite charges). Binding to the active site brings the substrate into close physical proximity, creating an enzyme-substrate complex. enzyme substrate enzyme-substrate complex J WERBA – IB BIOLOGY 8

ENZYME-SUBSTRATE SPECIFICITY The enzyme catalyses the conversion of the substrate into a product (or products), creating an enzyme- product complex. As the enzyme is not consumed in the reaction, it can continue to work once the product is completed. enzyme substrate enzyme-substrate complex enzyme product J WERBA – IB BIOLOGY 9

enzyme-substrate complex THE INDUCED-FIT MODEL U.1 The lock and key model of enzyme action does not fully explain enzyme activity. There are some enzymes that are capable of catalysing multiple reactions eg. some proteases have quite a broad specificity The Induced Fit Model proposes that the active site does not fit the substrate precisely until the substrate binds. enzyme-substrate complex enzyme product enzyme substrate J WERBA – IB BIOLOGY 10

THE INDUCED-FIT MODEL U.1 As the substrate binds to the active site, the active site changes shape to better fit the substrate. This weakens the bonds in the substrate, thus reducing the activation energy required for the reaction. J WERBA – IB BIOLOGY 11

ENZYME CATALYSIS U.2 Catalysis involves molecular motion and the collision of substrates with the active site. Collisions between the free moving enzymes and substrates are the result of random movements. Successful collisions will align the active site and substrate correctly, allowing binding to occur. J WERBA – IB BIOLOGY 12

ENZYME DENATURATION Enzymes, like all proteins, can become denatured. Denaturation is usually permanent. Can be caused by high temperatures and extremes of pH. Denaturation breaks bonds holding the 3D structure of a protein together  resulting in loss of shape/function J WERBA – IB BIOLOGY 13

ENZYME DENATURATION U.3 U.4 J WERBA – IB BIOLOGY 14

FACTORS AFFECTING ENZYME ACTIVITY U.3 U.4 J WERBA – IB BIOLOGY 15

FACTORS AFFECTING ENZYME ACTIVITY: Temperature Low temps result in insufficient thermal energy for the activation of a reaction to be achieved Increasing the temp 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 enzyme and substrate J WERBA – IB BIOLOGY 16

FACTORS AFFECTING ENZYME ACTIVITY: Temperature At an optimal temp, the rate of enzyme activity will be at its peak Higher temps will decrease enzyme stability, as the thermal energy disrupts the hydrogen bonds holding the enzyme together This causes the enzyme (particularly the active site) to lose its shape, resulting in a loss of enzyme activity (denaturation) J WERBA – IB BIOLOGY 17

FACTORS AFFECTING ENZYME ACTIVITY: pH U.3 U.4 Changing the pH will alter the charge of the enzyme, which in turn will change solubility and may change the shape of the molecule Changing the shape or charge of the active site will diminish its ability to bind to the substrate, abolishing enzyme function Enzymes have an optimum pH J WERBA – IB BIOLOGY 18

FACTORS AFFECTING ENZYME ACTIVITY: Substrate concentration Increasing substrate concentration will increase enzyme activity More substrate means there is an increased chance of enzyme and substrate colliding and reacting So more products will be formed in a given time period. After a certain point, the rate of reaction will stop rising despite further increases to substrate concentration, because as the environment has become saturated with substrate and all enzymes are in use J WERBA – IB BIOLOGY 19

FACTORS AFFECTING ENZYME ACTIVITY U.3 U.4 TEMPERATURE pH SUBSTRATATE CONCENTRATION Too low Inactivated (dormant) Denatured Enzyme efficiency is high Too high Enzyme efficiency plateaus J WERBA – IB BIOLOGY 20

ENZYME IMMOBILISATION U.5 Immobilised enzymes are widely used in industry. Methods: Aggregations of enzymes bonded together Attached to a surface – eg. glass Entrapped in gels – eg. alginate gel beads J WERBA – IB BIOLOGY 21

ENZYME IMMOBILISATION U.5 Immobilised enzymes are widely used in industry. Advantages: Enzyme is not dissolved – so concentration of substrate can be increased Enzymes can be recycled many times, saving time and money Collection of products is straight forward Increases enzyme stability to changing temp and pH J WERBA – IB BIOLOGY 22

ENZYME IMMOBILISATION U.5 Immobilised enzymes are widely used in industry. Examples: Used for medical diagnostic tests Used for food production – eg. lactose-free milk J WERBA – IB BIOLOGY 23

PRODUCTION OF LACTOSE-FREE MILK 70% of adult humans are lactose intolerant They have lost the ability to produce lactase in sufficient quantities after early childhood. A genetic mutation allows lactase production to continue through to adulthood. It is the absence of this mutation that results in lactose intolerance. J WERBA – IB BIOLOGY 24

PRODUCTION OF LACTOSE-FREE MILK Ingestion of milk will lead to excess gas production & diarrhoea. The food industry has produced lactose-free milk. Use lactase enzyme to break down lactose into glucose and galactose. Milk tastes sweeter but will not cause reaction in lactose-intolerant adults. J WERBA – IB BIOLOGY 25

PRODUCTION OF LACTOSE-FREE MILK Steps: Lactase is purified (from yeast or bacteria) Lactase is bound to an inert substance (such as alginate beads) Milk passed over immobilised enzymes becomes lactose-free J WERBA – IB BIOLOGY 26

PRODUCTION OF LACTOSE-FREE MILK Other uses: Source of milk for lactose- intolerant individuals Increases the sweetness of milk, thus reducing the need for artificial sweeteners Reduces the crystallisation of ice-creams Shortens the production time for yogurts or cheese J WERBA – IB BIOLOGY 27

ENZYMES Q1. The graph below shows the effect of substrate concentration on enzyme activity. What conclusion can be drawn about section X of the graph? The enzyme has started to denature and the reaction slows down. The reaction has finished and the substrate has been used up. The enzyme is saturated and is working at its maximum reaction rate. Some of the enzyme has been consumed and the reaction has reached a plateau. J WERBA – IB BIOLOGY 28

ENZYMES Q2. Which of the following will cause an enzyme to permanently lose its properties? I. Hydrolysis II. Freezing to –20°C III. Dissolving it in water I only II only I and II only I and III only J WERBA – IB BIOLOGY 29

ENZYMES Q3. What is decreased when lactase is added to milk? Sweetness Disaccharides Calcium Monosaccharides J WERBA – IB BIOLOGY 30

ENZYMES Q4. Discuss factors that affect enzyme activity. (9 marks) J WERBA – IB BIOLOGY 31

ENZYME EXPERIMENTAL DESIGN Catalase is an enzyme that catalyses the conversion of hydrogen peroxide into water and oxygen. Choose a research question and design an experiment to be run in class. Possible research Qs: What is the effect of substrate concentration? What is the effect of temperature? What is the effect of pH? Does the source of catalase influence the rate of reaction? J WERBA – IB BIOLOGY 32

ENZYME EXPERIMENTAL DESIGN Things you must consider about your IV: How are you going to vary your IV across a sufficient range? What units will you be measuring your IV in? Have you selected concentrations of chemicals that are safe to handle? J WERBA – IB BIOLOGY 33

ENZYME EXPERIMENTAL DESIGN Things you must consider about measuring your DV: Measuring increase of product or disappearance of substrate? Direct or indirect measurement? (eg. concentration or product or change in pH) Units and uncertainty of equipment? Time period needed? How many repeats are needed? J WERBA – IB BIOLOGY 34

ENZYME EXPERIMENTAL DESIGN Things you must consider about measuring your DV: J WERBA – IB BIOLOGY 35

ENZYME EXPERIMENTAL DESIGN Things you must consider about designing a fair test: What else could affect your DV? How/why would they affect your DV? How could you eliminate or reduce their influence on the DV? Do you need to monitor them throughout to be sure that they were controlled? Is there anything that was beyond your control? Safety and ethics of your method and equipment? J WERBA – IB BIOLOGY 36