Proteins and Enzymes

• Proteins:

  • all proteins contain the elements C, H, O and N
  • proteins are polymers made up of one or more chains of amino acid monomers known as polypeptides
  • Proteins have many functions, such as - structural, communication, enzymes, immune system and transport
• Amino acids:

  an amino acid consists of a central carbon atom with 3 side groups;

  • the amine group (NH2) - this is the basic part of the molecule
  • the carboxyl group (COOH) - this is the acidic group
  • the R group - this can be a variety of chemicals. Each amino acid has a different R group. There are around 20 different R groups
• Amino acid bonding:

  • amino acids can form chains by linking together
  • two amino acids join to form a dipeptide in a condensation reaction, releasing a molecule of water
  • the bond between the amino acids is called a peptide bond (strong covalent bond)
  • many amino acids can be linked together by condensation to produce a long chain polymer known as a polypeptide (protein)
• Primary structure:

  • this is the sequence of amino acids in a polypeptide chain. This order of amino acids is encoded in our DNA - if there is a mutation in the DNA you may get an incorrect primary structure, therefore the secondary, tertiary and quaternary structure may all be incorrect
  • the primary structure determines the shape of the whole protein
• Secondary structure:

  • this is the folding of the polypeptide chain. As polypeptide chains form they fold, allowing the formation of many weak hydrogen bonds
  • this produces a particular secondary structure, either the a-helix of the b-pleated sheet
• Tertiary structure:

  • this is the further folding where the whole chain (including secondary structures) folds into a specific 3D shape. This determines the proteins function (e.g enzyme’s activity sites)
• Bond in tertiary structure:

  Tertiary structure is stabilised by different bonds;

  • disulphide bonds (also known as disulphide bridges) - very strong covalent bonds between sulphur-containing amino acids, not easily broken down
  • ionic bonds - formed between the carboxyl and amino groups. They are moderately strong, but weaker than disulphide bonds. A change in pH can affect an ionic bond
  • hydrogen bonds - there are many of these however they are easily broken
• Quaternary structure:

  • this is found in proteins made of more than one polypeptide chain (e.g haemoglobin is made up of 4 polypeptide chains)
• Amino acid charges:

  • the primary structure is crucial to the shape of the protein - if it is incorrect, then it is possible incorrect bonds will form at each further level
  • some amino acids are positively charged , and some are negatively charged. The charge of the amino acid is important, as it is one of the factors that affect the bonds that can be made with other amino acids in the secondary, tertiary and quaternary layers
• Biuret test:

  • a simple biochemical test can be carried out to detect the presence of proteins in a solution
  • add potassium hydroxide and copper sulphate (or Biuret solution) to a sample of the solution to be tested
  • if protein is present then a lilac (purple) colouration is seen
  • if the sample does not contain a protein, only the blue colouration of the copper sulphate is seen (no colour change occurs)
• Enzyme theory:

  • all enzymes are globular proteins - they are soluble in water
  • all metabolic reactions in living organisms are catalysed by enzymes. Some of these are intracellular (act inside cells), other are extracellular (act outside cells)
  • enzymes are biological catalysts. A catalyst is a substance that increases the rate of a chemical reaction without being used up. They do not make a reaction happen that would not normally occur; they just speed up the rate it occurs
• Enzyme theory:

  • enzymes increase the rate of reaction by lowering the amount of activation energy needed to make the reaction to proceed. This is due to the fact that the enzyme will put pressure on the bonds, causing them to break with activation energy
  • without enzymes, the temperature in living cells (human body temperature, 37°C) would be too low for chemical molecules to react fast enough to support life
• Mechanisms of enzyme action:

  • the active site of an enzyme has a specific tertiary (3D) structure and shape. Enzymes usually only work on one type of substance (the substrate)
  • the substrate's molecular shape is said to be complementary to the enzyme's active site, which is where the substrate binds to
  • the enzyme combines reversibly with the substrate, which is held into the active site via temporary bonds, to form an enzyme-substrate complex
• The lock and key model:

  • this model suggests that the substrate combines with the enzyme at its active site in a precise way, like the way a lock and key fit together
  • the active site is always the exact complementary shape at the optimum temperature, so reactions are fastest in these conditions
  • one limitation is that this suggests that enzymes have a rigid structure. Scientists have since observed that enzymes are flexible
• The induced fit model:

  • this model suggests that when a substrate combines with an enzyme, it induces changes in the enzyme's structure. The active site chnages shape slightly to become fully complementary
  • the moulding of the active site around the substrate put strains on the bonds in the substrate , and so reduces the activation energy needed to break the bond
  • The induced fit model is a better explanation of enzyme action because it explains how the binding other molecules can affect the enzyme's shape and activity and provides an explanation for how the activation energy is lowered
• Enzymes in industry:

  • many of the reactions catalysed by enzymes have commercial uses. They are specific in their action and therefore less likely to produce unwanted by-products. They are biodegradable and so cause less environmental pollution
  • They work in mild conditions (low temperatures, neutral pH, normal atmospheric pressure) and are therefore energy saving. However, this can also been seen as a disadvantage as their conditions must be stringently controlled or the enzyme may become denatured
• Immobilised enzymes (commercial use):

  • Immobilised enzymes are enzymes that have been trapped into an inert matrix or material (alginate beads) which prevents them from moving during the reaction process
  • It makes recovery and reuse of the enzymes far more straightforward. This is particularly advantageous where the enzyme may be hard/expensive to produce
  • Immobilisation ensures the enzyme doesn't contaminate the final product, it allows for continuous processes and offers greater enzyme stability in variable or extreme temperatures or pH. This gives the reaction process greater efficiency
• Measuring Rate of Reaction:

  • you can measure the formation of products or the disappearance of the substrate
  • enzyme reactions produce a curve (the rate will be fastest at the start of the reaction). There are more substrate at the start, so more ES complexes are formed. As the reaction progresses, more substrate is used up, so less product is made
  • on a straight line pick a point on the line and divide the y-axis by the x-axis
  • on a curve, draw a tangent at a specific point. Make this into a right-angle triangle and divide the change in y by the change in x
• Low tempertaure affects enzyme action:

  • at very low temperatures enzyme molecules are inactive (they are not denatured)
  • at low temperatures molecules of enzyme and substrate have low kinetic energy, so move around slowly in aqueous solution. As they collide rarely, the rate of enzyme action remains low
• Enzymes optimum temperature:

  • as temperature increases, enzyme and substrate molecules gain more kinetic energy so move about quicker. As a result, they collide more frequently and with more force and so a greater number of successful collisions take place (more ES complexes are formed) so more product i made leading to an increased rate of reaction
  • Enzymes have an optimum temperature at which they work the fastest. For mammalian enzymes this is about 40°C. Other enzymes work better at different temperatures (snow flea enzymes work at -10°C, thermophilic bacteria enzymes work at 90°C)
• High temperature affects enzyme action:

  • at high temperatures, enzymes are denatured
  • the increased kinetic energy causes the hydrogen bonds holding the tertiary structure of the enzymes together to break. The active site changes shape so it is no longer complimentary to the substrate
  • enzyme-substrate complexes can no longer form. The rate of reaction drops as more enzymes become denatured
• Changes in pH affect enzyme action:

  • a change in pH alters the charges on the amino acids which make up the active site of the enzyme. This causes some of the hydrogen and ionic bonds that maintain the enzyme's tertiary structure to break and reform in different places
  • these changes alter the shape of the active site so it's no longer complimentary to the substrate. No more ES complexes can be formed, so the enzymes is denatured
• Enzymes optimum pH:

  • enzymes have an optimum pH at which they work the fastest. For most enzymes this is pH 7-8 (physiological pH of most cells), but few enzymes can work at extreme pH, such as protease enzymes in animal stomachs, which have an optimum of pH 2
  • pH fluctuations in organisms cells are relatively rare, so denaturing due to pH is unlikely. Small pH changes will reduce enzyme activity
  • a buffer solution can be used to control pH is an enzyme investigation. A buffer solution is used to produce a particular pH, and will maintain this pH if acids or alkalis are added
• The pH scale:

  • the symbol pH refers to the concentration of hydrogen ions (H+) present in a solution. The greater the concentration of H+, the lower the pH (more acidic)
  • a pH change of 1 changes the aqueous H+ concentration by 10 times (pH 2 contains 1000 times the concentration of aqueous H+ compared to an acid with pH 5)
  • the pH scale was created as a more convenient and manageable scale. (pH 1 has a 0.1mol dm -3 or 10 to the power of -1 concentration and pH 2 has 0.01mol dm-3 or 10 to the power of -2 concentration)
  • the pH scale is said to be logarithmic - pH = -log10 H+
• Substrate concentration affects enzyme action:

  • as the concentration of substrate increases the rate of reaction increases. This is because more substrate molecules means more collisions between the enzyme and substrate, so more ES complexes are formed (per minute/second)
  • the rate of reaction levels off. The limiting factor is the concentration of enzymes - even if more substrate is added the rate remains the same. This is because all the active sites are saturated so a maximum number of ES complexes are being made - the rate of reaction plateaus
• Enzyme concentration affects enzyme action:

  • as long as there is an excess of substrate, an increase in the amount of enzyme leads to a proportionate increase in the rate of reaction
  • if the substrate is limiting however, in other words there are not enough substrate molecules to occupy all the active sites at one time, then an initial increase in enzyme concentration will increase the rate of reaction, but eventually this levels off
• Enzyme inhibitors:

  • an inhibitor is a substance which decreases the rate of an enzyme controlled reaction by binding to the enzyme involved
  • there are 2 main types; competitive inhibitors and non-competitive inhibitors
• Competitive inhibitors:

  • competitive inhibitors have a similar shape to the substrate. This allows it to compete with the substrate - it can bind to the active site. Since the active site is blocked the substrate cannot bind to it, therefore the rate of reaction is reduced
  • the rate of reaction is reduced at low substrate concentrations. As the concentration increases the effect of inhibition decreases. High substrate concentration results in the same maximum rate being reached as with no inhibition (increased chance of occupying the active site)
• Non-competitive inhibitors:

  • these molecules do not combine with the active site (not a similar shape to the substrate). They bind to another region of the enzyme (allosteric site). This changes the tertiary structure of the enzyme so changes the active site shape . This results in fewer ES complexes so the rate of reaction decreases
  • the effect of a non-competitive inhibitor isn't dependent on it's concentration relative to the substrate (it doesn't compete)
  • The inhibitor inactivates the enzyme. A few enzymes may remain unaffected, so the reaction rate may proceed at a slow rate