Enzymes What are they? Globular Proteins: This is important in explaining how heat can denature them think tertiary structure Biological catalysts: They make reactions happen faster than they normally would at any given temperature. N.B. They do not react with the substrate. Reactions without enzymes Bonds broken: needs energy New bond made: releases energy Activation energy Energy used to break bonds of original molecule(s) Energy released in the formation of new bonds Where does activation energy come from? The energy needed to break the original bonds is kinetic (movement) energy. Kinetic energy of molecules is what we normally call heat energy. When molecules collide this kinetic energy is used to break the bonds. BANG Enzymes seem to reduce activation energy (1) Enzymes seem to reduce activation energy (2) Substrate molecules bind to the enzyme during the reaction. The enzyme allows the bond breaking to happen in small stages (an alternative pathway) rather than all at once in a big bang. The activation energy needed for each of these small stages is less than is needed for the whole reaction to happen at one go. So at any particular temperature (remember it is heat that supplies the energy) more of the substrate molecules will have enough energy to react. The overall effect of the enzyme is to seem to lower the activation energy necessary for the reaction to take place What happens during the reaction? Enzyme and substrate collide and fit together because they have complementary shapes The products do not have the exact same shape as substrate so do not fit the active site. They are released. Lock and key hypothesis This is the idea that the enzyme and substrate have complementary shapes. This explains why enzymes are very specific. E.2.1. Lock and key hypothesis Important aspect of enzyme action is its specificity. Two theories explaining why enzymes are specific. First theory proposed by Emil Fischer in 1890, lock and key hypothesis. Enzyme (lock), has unique shape complementary to substrate (key). Only when the two fit can substrate bind to enzyme. This gives enzyme its specificity. Hypothesis views enzymes as rigid, having fixed shape of active site (keyhole). Fischers lock and key hypothesis E.2.1. Lock and key hypothesis Once substrate entered active site and is changed into products, it no longer fits into active site. Products leave active site of enzyme allowing another substrate molecule to bind with empty active site. Induced Fit We now know that the lock and key hypothesis is not quite all there is to it. In the induced fit hypothesis the enzyme actually moulds itself around the substrate making the fit better still. The straining this causes to the substrate also begins the process of breaking bonds. E.2.2. Induced fit hypothesis In 1959, modification to lock and key hypothesis proposed by Daniel Koshland. This was induced fit hypothesis. Active site flexible physically; does not initially exist in shape complementary to substrate. Enzymes close up and enfold substrate when suitable substrates bind to binding residues of active sites. E.2.2. Induced fit hypothesis Substances too large or too small unable to fit into active site thus maintaining specificity of enzyme. Competitive inhibitors / very small molecules, though able to enter active site, cannot cause change in shape in site required for catalysis. Learning objective (a) Summary so far Enzymes are proteins They are specific They are catalysts (do not react) They work by breaking the reaction down into smaller steps (alternative pathway) This alternative pathway requires less activation energy at each stage than the whole reaction would. In the presence of an enzyme more reactions can happen at any particular temperature since a greater proportion of molecules will have the necessary activation energy In the reaction enzyme + substrate collide The substrate fits into the active site (determined by the tertiary structure of the enzyme). Also fit may be induced. An enzyme substrate complex is formed. The products of the reaction are released since they have a slightly different shape to the substrate molecules. Rates of Enzyme Catalysed Reactions 1. Temperature. E.4.3. Temperature At near or below freezing point, enzymes inactivated. When temperature increases, rate of enzyme-catalysed reaction increases. Due to kinetic energy of enzyme and substrate molecules becoming higher. More successful collisions between them causing more enzyme-substrate complexes to form, leading to greater rate of reaction. E.4.3. Temperature However, rate of reaction can only increase up to maximum level called optimum temperature. Further increases in temperature leads to breakage of bonds that hold enzyme (protein) in its 3-D shape. Bonds easily disrupted at higher temperatures by excessive molecular motion Hydrogen bonds especially sensitive to temperature changes. E.4.3. Temperature Enzyme denatured and no longer effective in catalysing reactions. Shape of active site changed and substrate no longer fits into it. E.4.3. Temperature Summary - Temperature speeds up the reactions because: The enzyme and substrate molecules have more kinetic energy so will move faster and collide more often (more reactions per second), and A greater number of substrate molecules will possess the necessary activation energy, but.. At high temperatures the bonds holding together the tertiary structure of the protein are shaken apart- denaturation. This changes the shape of the enzyme. Without the necessary active site shape substrate and enzyme cant bind together. 2. Concentration of Substrate E.4.2. Substrate Concentration Explanation: At low [S], rate of reaction increases with [S]. Many enzyme molecules have active sites unoccupied, and limited substrate molecules determines reaction rate. Increasing [S] increases reaction rate as more active sites used. More often, substrates collide successfully with enzyme, and rapidly converted to products. E.4.2. Substrate Concentration Up to certain [S], active sites fully occupied Rate of reaction becomes constant. Excess substrate molecules queuing for vacant active sites. Increase in rate achieved by increasing amount of enzyme. An increase in substrate concentration speeds up the reaction because : With more substrate molecules you will get a greater number of collisions between enzyme molecules and substrate molecules per second, but When all the active sites of the enzymes are fully occupied, an increase in substrate concentration no longer speeds up the reaction. The substrate is effectively queued up waiting for the next available active site. E.4.1. Enzyme concentration When enzyme concentration increases, rate of reaction increases proportionally to increase in enzyme concentration. Because more active sites available for substrates to react in. Rate of enzyme reaction Enzyme concentration Low enzyme concentration High enzyme concentration E.4.1. Enzyme concentration If amount of substrate is limited, the graph tapers off When substrate concentration limited, point reached when increasing enzyme concentration no longer has effect as there are many more empty active sites than substrates. Rate of enzyme reaction Enzyme concentration There is excess substrates and rate of reaction continues to increase. High enzyme concentration, limited substrate E.4.4. pH Enzymes operate optimally over very narrow pH range. As acidity increases or decreases, number of H + ions increase or decrease. This will disrupt ionic bonds within enzymes, altering conformation of enzyme. Shape of active site changed leading to decrease in enzyme efficiency. Fig 59. Effect of pH on the rate of reaction of different enzymes Inhibition: a) competitive Active site is blocked by the competitive inhibitor. For this to happen the competitor molecule needs to have a similar shape to the actual substrate molecule. If you increase the ratio of substrate to inhibitor you make it more likely that an enzyme- substrate complex will form rather than an enzyme inhibitor complex. This will reduce the effect of the inhibitor. Non-competitive inhibition Non-competitive inhibitor binds to a site other than active site. This causes a shape change in the whole enzyme molecule including the active site. Substrate can no longer bind. Increasing substrate concentration will not reverse this effect. E.6. Allosteric Inhibitors/Activators In most cases, molecules that affect enzyme activity bind to an allosteric site. A specific receptor site remote from active site. Most enzymes having allosteric sites are proteins with two or more polypeptide chains/subunits. Each subunit has its own active site and allosteric sites, usually located where subunits join. E.6. Allosteric Inhibitors/Activators Allosteric inhibitors have ability to change efficiency of enzymes. They attach to allosteric site away from active site and causes change in conformation of active site. Substrate can no longer bind with enzyme.