Enzymes and Their Applications PDF

PUBLIC / CYHOEDDUS Enzymes and their applied biological functions and applications  PUBLIC / CYHOEDDUS Learni...

PUBLIC / CYHOEDDUS Enzymes and their applied biological functions and applications  PUBLIC / CYHOEDDUS Learning Outcomes Describe the key principles of thermodynamics Understand how thermodynamics affects the different types of chemical bonds that can be formed between molecules Understand how enzymes lower the activation barrier of a reaction so it can occur Describe what enzymes are Describe the two theories of how enzymes work Understand the local factors which can affect enzyme activity Describe how competitive inhibitors of enzymes work Describe how non-competitive inhibitors of enzymes work Understand the different ways in which enzyme behaviour can be regulated Describe industrial and medical uses for enzymes  PUBLIC / CYHOEDDUS Principles of thermodynamics Most chemical reactions occur spontaneously A+B C Exergonic – release free energy +Substrates D How fast this reaction occurs Products depends on several key factors:- Concentration of substrate(s) available Temperature How many collisions will occur Most of these reactions are reversible Ultimately reach equilibrium Both reactions are occurring at the same rate  PUBLIC / CYHOEDDUS Biology is NEVER that neat!!!! Living cells never actually reach equilibrium In more elaborate systems, multiple connected reactions may occur This function makes it possible for the cell to have a dynamic metabolism, routing chemicals smoothly through the cell’s metabolic pathways.  PUBLIC / CYHOEDDUS What’s the difference? Enzymes Macromolecules Catalyse reactions without being used in the reaction Lower the activation barrier Biologically most enzymes are proteins Essential in metabolism otherwise most reactions would be to slows!!  PUBLIC / CYHOEDDUS How are enzymes able to do this? Proteins, DNA and other complex molecules of the cell are rich in free energy and have the potential to decompose spontaneously They persist because at cellular temps most can’t move past the Ea  PUBLIC / CYHOEDDUS Activation of the Atoms settle into new stable reactants states An enzyme cannot change the ΔG for a reaction. I.e. it Sig. of 0 is cannot make an endergonic that reaction exergonic. reaction is Enzymes can only speed exergonic reactions up that would and eventually occur anyway. spontaneo Enzymes are very specific us for the reactions they catalyse, they determine which chemical processes will be going on in the cell at any particular time.  PUBLIC / CYHOEDDUS Naming of enzymes Most enzyme names end with the suffix –ase Suffix attached to the name of their substrate or a descriptive term for reactions they catalyse e.g. Urease has urea as a substrate whilst alcohol dehydrogenase catalyses the removal of hydrogen from alcohols (i.e. oxidation of alcohols) Exceptions Some known by the historic names e.g. trypsin and amylase Some named after genes e.g. recA gene and HSP70 (Heat shock protein) are both enzymes that catalyse the hydrolysis of ATP  PUBLIC / CYHOEDDUS Enzymes Most prominent catalysts of biological systems Metabolic processes essential to life could not continue without enzymes Act as controls as well as affect pace of reactions As catalysts they enhance the rate of reaction without being permanently affected themselves Can be temporarily altered during the process but remains unchanged overall in the reaction Enzyme catalysed reactions occur 103 – 1020 times faster than the corresponding uncatalysed reactions Extremely efficient and specific for both reaction and substrate  PUBLIC / CYHOEDDUS What do they do? Most enzymes are a specialised form of globular protein Enzymes are specific to a particular substrate or group of substrates Only the active site is involved with binding to the substrate Substrate binding to enzyme is a rapidly reversible process Reaction occurs and product(s) dissociate No formation of waste products  PUBLIC / CYHOEDDUS Specificity Intimate fit between enzyme and substrate Many enzymes exhibit stereospecificity - only act on a single stereoisomer of the substrate  PUBLIC / CYHOEDDUS Induced Fit Theory Only a restricted region of the enzyme molecule actually binds to the substrate This active site is typically a pocket or groove (formed of only a few amino acids) on the surface of the enzyme where catalysis occurs Enzyme specificity is because of a compatible fit between active site and substrate shapes  PUBLIC / CYHOEDDUS Induced fit model under the microscope As the substrate enters the active site, the enzyme changes shape due to interactions between the substrate’s chemical groups and chemical groups on the side chains of the amino acids forming the active site. This induced fit is like a clasping handshake. Induced fit brings chemical groups of the active site into positions that enhance their ability to catalyze the chemical reaction.  PUBLIC / CYHOEDDUS Specificity and catalysis The rate at which a particular amount of enzyme converts substrate to product is partly a function of the initial concentration of the substrate. I.e., the more substrate molecules that are available, the more frequently they access the active sites of the enzyme molecules. But, there is a limit to how fast the reaction can be pushed by adding more substrate to a fixed concentration of enzyme. At some point, the concentration of substrate will be high enough that all enzyme molecules have their active sites engaged. Now the enzyme is said to be saturated, and the rate of the reaction is determined by the speed at which the active site converts substrate to product.  PUBLIC / CYHOEDDUS Factors affecting enzyme activity 1. Temperature Maximum rate of reaction at optimum temperature Denatured at higher values Different for each enzyme usually reflecting environment of source organisms 2. pH Changes affect ionisation of amino acids causing changes in 3D shape Leads to a reduction in catalytic properties  PUBLIC / CYHOEDDUS Factors affecting enzyme activity 3. Cofactors and/or coenzymes Can be permanently or reversibly bound to the enzyme. Cofactors – non-protein components e.g. Metal ions such as Mg2+ or Zn2+ Coenzymes – organic molecules which function like a second substrate briefly associating with the enzyme e.g. NAD+/NADH or vitamin precursors such as Vitamin B1 (thiamine)  PUBLIC / CYHOEDDUS Respiration, enzymes and temperature Ectothermic animals Have a wide variations in body temperature during a 24hr period Q10 law Rate of respiration and therefore metabolic rate is roughly twice as fast at 30°C than at 20°C Such organisms are only active when their body temperature allows them to react and move quickly  PUBLIC / CYHOEDDUS So what might affect enzyme kinetics? Low [S] At any instant only a small fraction of the enzyme molecules have substrate bound As [S] increases Fraction of enzyme molecules bound to substrate increases High [S] Most enzyme molecules have substrate bound – results in saturation  PUBLIC / CYHOEDDUS Enzyme Inhibitors Substrate binds to active site using induced fit mechanism Ea of catalysed reaction is reduced Reaction takes place much quicker without enzyme consumption  PUBLIC / CYHOEDDUS Competitive inhibition Many enzyme inhibitors bind to the enzyme by weak interactions Mimic the substrate Compete for the active site Many enzyme inhibitors bind to the enzyme by weak interactions This inhibition is reversible  PUBLIC / CYHOEDDUS Overcoming reversible competitive inhibition Overcome by increasing the concentration of substrate This way, as active sites become available, there’s more enzyme than inhibitor to gain entry to the active site  PUBLIC / CYHOEDDUS Non-competitive inhibition Impede enzymatic reactions by binding to another part of the enzyme Changes the shape of the enzyme, making the active site less effective Therefore makes the enzyme less effective at catalysing the conversion of substrate to product  PUBLIC / CYHOEDDUS Bio-weapon vs Therapeutic drug To prevent over-excitation of muscle cells, enzyme AChesterase removes excess Ach from the synaptic cleft VX nerve agent irreversibly inhibits ACh-esterase so ACH is not cleared from the cleft resulting muscle seizures and death results very quickly Neostigmine reversibly inhibits ACh-esterase and can be used therapeutically in myasthenia gravis treatment Prolonged ACh activity is benefical  PUBLIC / CYHOEDDUS Allosteric Activation If all the cell’s metabolic pathways operated simultaneously, there’d be cellular chaos It is essential for life that the cell can tightly regulate its metabolism, controlling when and where reactions take place by controlling when and where reactions take place by controlling when and where the enzymes are active Achieved by switching on and off genes that encode specific enzymes which regulate those enzymes’ activity once they’re made  PUBLIC / CYHOEDDUS Allosteric activators and inhibitors Allosteric regulation is defined as “Any case in which a protein’s function is affected by the binding of a regulatory molecule to a separate site” Results in inhibition or stimulation of enzymatic activity Most allosteric enzymes are composed of two or more subunits, each with its own active site The entire complex spends its time oscillating between two shapes, one catalytically active and one inactive  PUBLIC / CYHOEDDUS Allosteric activators and inhibitors In the simplest kind of allosteric action, an activating or inhibiting regulatory molecule binds to a regulatory site The binding of an activator stabilises the shape that has functional active sites and vice-versa The sub-units of an allosteric enzyme fit together in such a way that a shape change in one sub-unit is transmitted to all others So a binding of a single activator or inhibitor molecule to one regulatory site will inactivate all the active sites of all the sub-units  PUBLIC / CYHOEDDUS Co-operativity Inactive Active state state A substrate molecule binding to one active site in a multi-subunit molecule triggers a shape change in all the sub-units Increases activity in other active sites Mechanism amplifies the response of enzymes too substrates Example: The binding of oxygen to haemoglobin  PUBLIC / CYHOEDDUS Feedback inhibition Product formed in the reaction stops the enzyme from functioning IS NOT a form of competitive inhibition The product does not have the same shape as the substrate for the active site ATP allosterically inhibits enzymes in an ATP generating pathway using this common metabolic control mechanism Metabolic pathway is switched off by the inhibitory binding of its end product to an enzyme that acts earlier in the pathway  PUBLIC / CYHOEDDUS Specific localisation The cell is compartmentalised Structures bring order to the metabolic pathways Arrangement facilitates a sequence of reactions with the end product from the first enzyme becoming the substrate for the next enzyme in the complex and so on ….  PUBLIC / CYHOEDDUS Medicine Analytical test: Diabetics use test strips of paper impregnated with glucose oxidase to monitor their blood sugar Presence of the enzyme where it should not be can help diagnose disease e.g. in liver disease enzymes leak into the bloodstream Therapeutic enzymes: replace enzymes that are missing (deficient) e.g. clotting factors to treat haemophilia or proteases are used to degrade fibrin to prevent the formation of dangerous blood clots  PUBLIC / CYHOEDDUS Summary Rate of a chemical reaction depends on the concentrations of the reactants The higher the concentration the faster the reaction Almost all biochemical reactions are reversible When the forward and reverse reactions are equal, the reaction is at equilibrium Enzymes are essential biological catalysts most of which are globular proteins Enhance the rate of reaction without being permanently affected themselves Only the active site is involved in the reaction process though enzyme activity can be affected by temperature, pH and the presence of co-factors or co-enzmyes Targeting of enzyme activity can be achieved using competitive or non-competitive mechanisms Enzyme activity can be regulated by several means within a cell including:- Allosteric Compartmentalisation Feedback inhibition Co-operativity