Biology module 2.4

• Enzymes


  Increase the rate of reaction by lowering the activation energy of the reaction they catalyse
• Active site


  The area of the enzyme where the reaction with the substrate takes place
• Enzymes


  • Specific to substrates they bind to, meaning that only one type of substrate fits into the active site of the enzyme
  • When the enzyme and substrate form a complex, the structure of the enzyme is altered so that the active site of the enzyme fits around the substrate (induced fit model)
• Factors affecting the rate of enzyme-controlled reactions

  • Enzyme concentration
  • Substrate concentration
  • Temperature
• Enzyme concentration


  The rate of reaction increases as enzyme concentration increases as there are more active sites for substrates to bind to, however increasing the enzyme concentration beyond a certain point has no effect on the rate of reaction as there are more active sites than substrates so substrate concentration becomes the limiting factor
• Substrate concentration


  As concentration of substrate increases, rate of reaction increases as more enzyme-substrate complexes are formed. However, beyond a certain point the rate of reaction no longer increases as enzyme concentration becomes the limiting factor
• Temperature


  Rate of reaction increases up to the optimum temperature, which is the temperature at which enzymes work at their maximum rate. Rate of reaction decreases above the optimum temperature
• Inhibitors


  Substances which slow down or stop a reaction by affecting the binding of substrate to the enzymes
• Types of inhibitors


  • Reversible
  • Irreversible
• Irreversible inhibitors


  • Heavy metal ions such as mercury and silver
  • Cyanide
• Irreversible inhibitors


  • Cause disulphide bonds within the protein structure to break, as a result causing the shape of the active site to change, thus affecting protein activity
  • Covalently bind to the active site, therefore preventing the binding of the substrate
• Reversible inhibitors


  Bind to the active site through hydrogen bonds and weak ionic interactions, therefore they do not bind permanently
• Types of reversible inhibitors


  • Competitive
  • Non-competitive
• Competitive inhibitors


  Similar in structure to the substrate molecule, therefore they bind to the active site of the enzyme, decreasing its activity as they compete with substrate for the enzyme. The amount of product formed remains the same, however the rate at which product formation occurs decreases. Increasing the substrate reverses the effect of competitive inhibitors by outcompeting them
• Non-competitive inhibitors


  Do not bind to the active site; they bind at another site on the enzyme known as the allosteric site. Binding of the non-competitive inhibitors changes the shape of the active site therefore preventing the binding of the substrate. Increasing the concentration of substrate has no effect on non-competitive inhibition
• Drugs that are inhibitors

  • Penicillin (inhibits enzyme transpeptidase which plays an important role in cell wall formation)
  • Ritonavir (inhibits HIV protease which is responsible for assembly of new viral particles and spread of infection)
• Cofactors


  Non-protein compounds required for the enzyme's activity to occur
• Types of cofactors


  • Coenzymes
  • Activators
  • Prosthetic groups
• Coenzymes


  Organic cofactors which do not bind permanently, and facilitate the binding of substrate to enzyme. Many are vitamin derived, e.g. NAD derived from niacin, which acts as a hydrogen acceptor
• Activators


  Inorganic metal ions which temporarily bind to the enzyme and alter its active site, making the reaction more feasible. For instance, magnesium ion is an important activator which is involved in processes such as shielding negative charge
• Prosthetic groups


  Permanently attached to the enzyme. For instance, haemoglobin contains a prosthetic haem group which contains iron, permanently bound to the molecule, which serves as a means of binding oxygen
• Enzymes


  Biological catalysts that speed up the rate of chemical reactions without being used up or undergoing permanent change
• Enzymes


  • They are globular proteins with complex tertiary structures
  • Some are formed from a single polypeptide, whilst others are made up of two or more polypeptides and therefore have a quaternary structure
• Intracellular enzymes


  Enzymes produced and functioning inside the cell
• Extracellular enzymes


  Enzymes secreted by cells and catalysing reactions outside cells (e.g. digestive enzymes in the gut)
• Extracellular digestive enzymes


  • Amylase (hydrolyses starch into maltose)
  • Trypsin (breaks proteins down into peptides and amino acids)
• Some organisms can only feed using a form of extracellular digestion in which the digestive enzymes are secreted outside of their bodies
• Active site


  Where specific substrates bind forming an enzyme-substrate complex
• Enzyme specificity


  The complementary nature between the shape of the active site on the enzyme and its substrate(s)

  
• Enzyme-substrate complex


  Formed when an enzyme and its substrate join together temporarily before the enzyme catalyses the reaction and the product(s) are released

  
• Lock-and-key hypothesis


  Enzymes and substrates are rigid structures that lock into each other very precisely
• Induced-fit hypothesis


  The enzyme and its active site (and sometimes the substrate) can change shape slightly as the substrate molecule enters the enzyme, maximising the ability of the enzyme to catalyse the reaction

  
• Activation energy


  The amount of energy needed by the substrate to become just unstable enough for a reaction to occur and for products to be formed
• Enzymes


  Speed up chemical reactions by providing an alternative energy pathway with a lower activation energy
• Optimum pH


  The pH at which an enzyme operates best
• Enzymes are denatured at extremes of pH
• Investigating the effect of pH on enzyme reaction rates


  1. Use buffer solutions to maintain a specific pH

  2. Add a measured volume of buffer solution to the reaction mixture

  3. Use iodine solution as an indicator for the amylase-catalysed breakdown of starch
• Investigating the effect of pH on enzyme activity


  1. Controlled and the effect of changing pH can be measured

  2. Mixing enables the enzymes and substrate to be equally mixed

  3. After 10 seconds, use a pipette to place one drop of the mixture on the first drop of iodine, which should turn blue-black

  4. This test indicates whether starch is still present

  5. Wait another 10 seconds and place another drop of the mixture on the second drop of iodine

  6. Repeat every 10 seconds until iodine solution remains orange-brown

  7. When the solution remains orange-brown it means amylase has broken down all of the starch so nothing is left to react with the iodine

  8. Repeat experiment at different pH values

  9. The less time the iodine solution takes to remain orange-brown, the quicker all the starch has been digested and so the better the enzyme works at that pH
• The above method can be adapted to control temperature by using a water bath at 35℃
• All solutions that need to be used (starch, amylase, pH buffers) should be placed in a water bath and allowed to reach the temperature (using a thermometer to check) before being used