Enzymes Presentation PDF

Enzymes What do I know already?  Objectives Define metabolism Describe the difference between solar and cellular energy Define the term enzymes Describe the structure and function of enzymes in plant and animal metabolism Describe the use of immobilised enzymes in bioproc...

Enzymes What do I know already?  Objectives Define metabolism Describe the difference between solar and cellular energy Define the term enzymes Describe the structure and function of enzymes in plant and animal metabolism Describe the use of immobilised enzymes in bioprocessing Prepare an enzyme immobilization and examine how it is applied Explain and investigate the effect of pH and temperature on the rate of enzyme activity Use the active site theory to explain the function and specificity of enzymes Define the term optimum activity in relation to pH Describe the nature and roles of ATP and NADP+ Describe heat denaturation of proteins and investigate the heat denaturation of one particular enzyme  Key Words ⚫ Metabolism ⚫ Solar ⚫ Cellular ⚫ Catalyst ⚫ Enzyme ⚫ Product ⚫ Substrate ⚫ Temperature ⚫ pH ⚫ Immobilization ⚫ Bioreactor ⚫ Bioprocessing  Metabolism ⚫ Metabolism is the sum of all the chemical reactions that take place in the body ⚫ E.g. growth, movement, maintenance of constant temperature, repair, responses, reproduction ⚫ Energy is either released or absorbed ⚫ Metabolism maintains a balanced internal state (homeostasis)  Sources of Energy ⚫ Solar Energy ⚫ Cellular Energy  Solar Energy ⚫ Primary source of energy on earth is sunlight ⚫ Some is trapped by organisms that contain pigments to absorb light e.g. chlorophyll ⚫ Producers use solar energy to form chemical bonds in carbohydrates ⚫ Conversion of solar energy to chemical energy in photosynthesis  Cellular Energy ⚫ Cellular energy is the energy stored in the bonds of bio-molecules (nutrients) ⚫ Energy is able to be released by reactions in a cell ⚫ Energy can be passed along a food chain from producers to consumers ⚫ Respiration releases energy when bio-molecules are broken down  Enzymes ⚫ Enzymes are catalysts made of protein ⚫ A catalyst is a substance that speeds up a reaction without itself being used up in the reaction  Features of Enzymes ⚫ Made of protein - long chains of amino acids ⚫ Folded into a 3D shape ⚫ They work as they have the correct shape to fit the substrate they are acting on e.g. like a lock and key  Features of an Enzyme ⚫ Substrate – substance with which an enzyme reacts ⚫ Product – substance the enzyme forms Example: Amylase Starch Maltose (substrate) enzyme (product)  Features of Enzymes ⚫ Anything that changes the shape of an enzyme will reduce the efficiency of the enzyme ⚫ Enzyme shape can be changed by: ⚫ Temperature ⚫ pH  Feature of Enzymes ⚫ Enzyme reactions are reversible ⚫ Enzymes can be anabolic – form complex compounds ⚫ Enzymes can be catabolic – break down large compounds  Role of Enzymes ⚫ Control metabolic reactions in plants and animals ⚫ Catabolic enzymes ⚫ Anabolic enzymes  Catabolic Enzymes ⚫ Amylase – converts starch to maltose ⚫ Breaks down a substance to smaller parts ⚫ Made by the salivary glands and the pancreas to aid digestion ⚫ When seeds germinate amylase converts starch to maltose ⚫ Lipase and pepsin are catabolic enzymes ⚫ Enzymes used in respiration  Anabolic Enzymes ⚫ DNA ligase used in genetic engineering to join two pieces of DNA together ⚫ DNA polymerase forms and repairs DNA ⚫ Convert simpler molecules to a more complex form ⚫ Enzymes in photosynthesis – convert water and carbon dioxide to glucose  Factors Affecting Enzyme Activity ⚫ Enzymes work best under ideal conditions ⚫ Change in these alters the rate of reactivity ⚫ Changes include: ⚫ Temperature ⚫ pH ⚫ A denatured enzyme is an enzyme that has lost its shape and can no longer function  Temperature ⚫ At low temperatures cell contents become solid and enzymes cannot work ⚫ When temperature increases the rate of molecular movement increases ⚫ Substrate molecules and enzymes collide increasing the rate of reaction  Temperature ⚫ Human enzyme work best at 37oc ⚫ Plant enzyme work best between 20oc and 30oc ⚫ Above certain temperature enzymes will loose their 3D shape and rate of reaction fall again ⚫ Denaturation takes place and the enzyme no longer functions  pH ⚫ pH scale 0 – 14 ⚫ 1 to 7 is acidic, 7 is neutral, 7 to 14 is basic ⚫ Enzymes work over a narrow range of pH ⚫ Mostly pH range is between 6 and 8 – optimum pH is 7 ⚫ Outside this range the enzyme loses its shape - denatured ⚫ Pepsin in the stomach will function at a pH of 2  Note ⚫ Enzyme activity is also affect by: ⚫ Enzyme concentration ⚫ Substrate concentration  Immobilised Enzymes ⚫ Bioprocessing is the use of enzyme controlled reactions to create a product ⚫ E.g. antibiotics, vaccines, food colouring and flavourings, vitamins, perfumes ⚫ The use of micro-organisms to create a product has been replaced by purified enzymes ⚫ A bioreactor is a vessel or container in which living cells or their products are used to make a product  Immobilised Enzymes ⚫ Adding enzymes freely to a bioreactor is wasteful ⚫ It is not possible to isolate the enzymes from the product for reuse ⚫ Enzymes can be fixed or immobilised so they are easier to recover and reuse ⚫ Immobilised enzymes are enzymes that are attached or fixed to each other or to an inert material  Methods of Immobilising Enzymes 1. Attached to each other 2. Attached to insoluble supports 3. Enclosed with a membrane or gel  Methods of Immobilising Enzymes Chemical Methods Physical Methods Enzymes are bonded to Adsorption – enzymes are each other physically attached to an inactive support e.g. glass beads, cellulose particles Enzymes are bonded to an Enclosed in a membrane inactive (inert) support Enclosed in a gel (sodium alginate) – the substrate is able to enter and exit the gel Adsorption Enclosed by a membrane Enclosed in a gel  Bonded to each Bonded to an inert other support  Advantages of Immobilised Enzymes 1. They can be reused – cost of replacements can be high 2. Remain in the bioreactor at the end of the process – no need for separation 3. Increases the stability of the enzyme 4. Production process is cheaper  Uses of Immobilised Enzymes ⚫ Sweeten soft drinks with fructose Glucose Isomerase Glucose Fructose ⚫ Develop new antibiotics Penicillin Acylase Penicillin New Antibiotic ⚫ Replace condensed milk when making toffee and caramel Lactase Lactose Glucose + Galactose  Enzyme Experiments – varying the 4 factors in enzyme experiments Factor Method to keep Method used to factor constant vary factor Temperature Use water baths at the Use water baths at same temperature different temperatures pH Use the same pH Use pH buffers of buffer different values Enzyme concentration Add the same volume of enzyme Substrate Add equal volumes of concentration the same substrate Extended Study of Enzymes and Energy Carriers Enzyme Action  No longer the Lock and Key principle but can be described as a Bean Bag fit What happens when you sit on a bean bag????  Active Site ⚫ The active site is the part of an enzyme that combines with the substrate ⚫ Not a rigid shape – it is a depression on the surface of the enzyme ⚫ Enzyme made of protein with a 3D shape ⚫ 2 or more globular sections called domains joined together ⚫ Active site larger than the substrate but it is specific to the substrate it acts on Active Site Location 2 Globular Sections join together to create the Enzyme  Active Site ⚫ Changes shape slightly when it comes in contact with the substrate ⚫ Fits more precisely around the substrate ⚫ Induced fit model or the active site theory ⚫ E.g. bean bag effect! Recognise the difference between A and B B is more accurate for explaining Enzyme Specificity  Active Site Theory 1. Substrate combines with the 3D active site of the enzyme 2. Active site changes shape slightly (induced) 3. An enzyme – substrate complex is formed 4. The bonds in the substrate are altered so it changes into the product(s) 5. Product leaves the active site 6. Returns to its original shape and can accept a new substrate molecule  1. Substrate combines with active site Active Site Enzyme Substrate 2. Active site changes shape slightly Active Site Enzyme Substrate 3.Enzyme – Substrate complex form 4. Bonds are broken Substrate changed to Enzyme Substrate products which are complex released  5. Products are released and enzyme returns to original shape Products Active Site Enzyme New Substrate Induced Fit Theory Animation  Enzyme Specificity ⚫ Enzyme specificity means that each enzyme will react with only one particular substrate ⚫ If the active site shape is changed the enzyme’s ability to function is reduced  Optimum pH ⚫ Active site is sensitive to pH ⚫ Enzymes are adapted to function at a certain pH ⚫ E.g. amylase pH 7, pepsin pH 2 ⚫ If pH is unsuitable the active site will change shape and no longer accept the substrate ⚫ Enzymes have optimum activity at specific pH values ⚫ An enzyme’s optimum pH means the pH value at which the enzyme works best A change in pH affects the Active Site Animation  Enzymes and Temperature ⚫ Less sensitive to changes in temperature than pH ⚫ Increasing temperature increases molecular movement ⚫ Enzymes collide more frequently with their substrates ⚫ Rate of reaction increases as temperature increases ⚫ However, Above a certain temperature active site will begin to lose shape  Denaturation ⚫ A denatured enzyme has lost its shape and can no longer carry out its function ⚫ Caused by: ⚫ High temperature ⚫ pH values outside the optimum pH ⚫ Chemical and radiation ⚫ Protein loses its 3D shape and the active site loses the ability to react with the substrate ⚫ Permanent or irreversible process  Energy Carriers Chemical Reactions: ⚫ Photosynthesis – energy from sunlight used to make food ⚫ Respiration – food broken down to release energy ⚫ ADP and ATP store and transfer energy in these reactions ⚫ NAD+ and NADP+ trap and transfer electrons and hydrogen ions in cell reactions ADP and ATP  ADP ⚫ Adenosine Diphosphate ⚫ Made of the base adenine, a 5 carbon sugar called ribose and 2 phosphate groups ⚫ Bond between the phosphate groups is unstable ⚫ It is a low energy molecule  ATP ⚫ Adenosine Triphosphate ⚫ Forms when a third phosphate group is added to ADP ⚫ Extra energy is also added with the extra bond ⚫ Process of adding another phosphate is called phosphorylation ⚫ High energy compound which can move around in cells to carry energy ⚫ Energy cannot be stored, must be used immediately  ATP ⚫ Breaks down to release energy and a phosphate group ⚫ Reforms ADP ⚫ In respiration breakdown of glucose forms ATP which provides energy for movement, protein production, urine formation etc…  Energy Carriers Chemical Reactions: ⚫ Photosynthesis – energy from sunlight used to make food ⚫ Respiration – food broken down to release energy ⚫ ADP and ATP store and transfer energy in these reactions ⚫ NAD+ and NADP+ trap and transfer electrons and hydrogen ions in cell reactions  ADP ⚫ Adenosine Diphosphate ⚫ Made of the base adenine, a 5 carbon sugar called ribose and 2 phosphate groups ⚫ Bond between the phosphate groups is unstable ⚫ It is a low energy molecule  ATP ⚫ Adenosine Triphosphate ⚫ Forms when a third phosphate group is added to ADP ⚫ Extra energy is also added with the extra bond ⚫ Process of adding another phosphate is called phosphorylation ⚫ High energy compound which can move around in cells to carry energy ⚫ Energy cannot be stored, must be used immediately  ATP ⚫ Breaks down to release energy and a phosphate group ⚫ Reforms ADP ⚫ In respiration breakdown of glucose forms ATP which provides energy for movement, protein production, urine formation etc…  Formation of ATP ADP + energy + P ATP + Water Breakdown of ATP ATP + Water ADP + energy + P  + NADP and NADPH  NADP + ⚫ Nicotinamide adenine dinucleotide phosphate ⚫ Low energy molecule used in photosynthesis ⚫ Hydrogen atom contains a proton (H +) and an electron ⚫ NADP + can accept a pair of high energy electrons with a proton to form NADPH ⚫ The addition of electrons to a molecule is called reduction ⚫ NADP + is reduced to NADPH  NADPH ⚫ An electron and hydrogen carrier ⚫ Used to form glucose in photosynthesis ⚫ Releases 2 high energy electrons and a hydrogen ion or proton when it breaks down  Formation of NADPH + from NADP NADP + + 1e- NADP NADP + 1e- NADP- - + NADP + H NADPH NADP + +2 electrons + H+ NADPH (low energy) (high energy)  Breakdown of NADPH NADPH NADP- + 2 electrons + H+ (high energy) (low energy)  NAD + and NADH ⚫ NAD is used in respiration as a low + + energy molecule – equivalent to NADP ⚫ NADH is used as a high energy molecule – equivalent to NADPH  Summary Low Energy High Energy Photosynthesis ADP, NADP+ ATP, NADPH Respiration ADP, NAD+ ATP, NADH  Have We? Define metabolism Describe the difference between solar and cellular energy Define the term enzymes Describe the structure and function of enzymes in plant and animal metabolism Describe the use of immobilised enzymes in bioprocessing Prepare an enzyme immobilization and examine how it is applied Explain and investigate the effect of pH and temperature on the rate of enzyme activity Use the active site theory to explain the function and specificity of enzymes Define the term optimum activity in relation to pH Describe the nature and roles of ATP and NADP+ Describe heat denaturation of proteins and investigate the heat denaturation of one particular enzyme