Enzymes: Biological Catalysts

Enzymes

• Enzymes are biological catalysts, made of proteins, accelerating chemical reactions without being consumed.
• They are globular proteins, soluble in water.
• Dipping sliced apples in 70-80°C water for a minute prevents browning by denaturing enzymes.
• Polyphenols in apples bind with oxidizing enzymes, which act as locks and lead to browning.

Lock and Key Hypothesis

• Enzymes have an active site that is like a lock, where a substrate fits and binds like a key.
• The enzyme-substrate complex is formed when a substrate binds to an enzyme.
• Within this complex, new molecules (products) are formed.
• After the reaction finishes, the enzyme can bind to another substrate.
• An enzyme's active site is complementary to its substrate, meaning a specific enzyme acts on a particular substrate only.
• Amylase works on starch, protease on proteins, and lipase on fats.

Activation Energy

• Activation energy is required for chemical reactions to occur, as substrates need extra energy to convert to products.
• Benedict's test for reducing sugar uses heat to provide activation energy.
• Enzymes lower the activation energy of reactions by holding substrates in a way that molecules react more easily.
• Enzyme-catalyzed reactions happen faster and at lower temperatures.
• Enzymes do not alter the energy released in product formation, nor do they change the reaction's energy yield.

Active Sites

• Active sites result from the tertiary structure of proteins and form a unique 3D shape for each enzyme.
• The 3D shape creates pockets or grooves where the active sites are located.
• The arrangement of amino acids within the 3D shape makes active sites specific to particular substrates.
• 3D shape of enzyme determines active site formation and specificity.

Induced Fit Hypothesis

• The shape of the active site is not always static, which lead to the induced fit hypothesis
• The induced fit hypothesis is similar to the lock and key version, but the enzyme molecule is more flexible
• It states that the enzyme is flexible, with an active site that changes shape as the substrate approaches for a perfect fit.
• This is more efficient for catalysis.
• Temporary bonds hold the substrate to form an enzyme-substrate complex.
• This complex reduces the activation energy, allowing a faster reaction.
• Substrate converts to a product via R-groups in the active site.
• After completing a reaction, products move away from the enzyme, and the active site returns to its initial shape.

Lysozyme

• Lysozyme is an example of an enzyme that uses the induced fit hypothesis.
• It is found in tears, saliva, and other bodily secretions.
• Lysozyme has a tertiary level of protein structure, changing shape to fit its substrate.

Measuring Rate of Reaction

• The rate of a reaction can be measured through:
  • Measurement of product formation
  • Measurement of substrate disappearance
• The rate depends on the number of substrate molecules.
• It also depends on the enzyme's speed in converting substrate to the product and move on to other substrate molecules.

Measurement of Rate of Formation of Product

• More substrates that turn into products means fewer substrate molecules exist.
• The reaction slows down as fewer substrate molecules are left, eventually stopping.
• Measure the initial reaction rate by calculating the tangent's slope to the curve, close to time zero.
• Only measure the initial rate from the graph.
• Graph's increment phase: more substrates react with enzymes to form enzyme-substrate complexes.
• Decline phase: substrates are used up.
• The concentration of the enzymes do NOT deplete following a reaction.

Measurement of Rate of Disappearance of Substrate

• Use a colorimeter for measuring starch concentration.
• Prepare starch solution concentrations in test tubes.
• Add two drops of iodine solution to each test tube.
• Measure absorbance values using the colorimeter.
• Plot absorbance against concentration; the graph will show the standard curve.
• Undegraded starch and iodine solutions produce a blue-black colour.
• The intensity is detectable using a colorimeter.
• As the reaction progresses, the blue-black colour of samples decreases.
• Plotting starch concentration against time shows a gradual decline.
• The initial rate of starch disappearance can be found by the slope of the tangent at the reactions starting point.

Factors Affecting Enzyme Action

• Enzymes are controlled by factors affecting their actions:
  • Temperature
  • pH
  • Enzyme concentration
  • Substrate concentration
  • Inhibitor concentration

Temperature

• Low temperature: kinetic energy is low, lowering movement speed.
• The chance of product formation decreases, with rare binding between substrate and enzyme.
• Increased temperature: molecules gain kinetic energy, which results in more effective collisions.
• Effective collisions cause substrate molecules to react, resulting in product formation.
• Optimum temperature has the most collisions, and forms max product.
• Very high temperature: destabilizes enzymes' shape and structure, causing denaturation.
• Optimum temperature favors enzymes and reactions, unlike low and high temperatures.

pH

• Enzymes prefer a pH with the highest rate of activity, referred to as the optimum pH.
• pH alters active sites which cause denaturation to the enzyme.
• It affects the function of enzymes by influencing the structure.
• pH influences the bonds that stabilize the whole enzyme structure.
• High levels of hydrogen ions affect the R-groups of the amino acids of the active site.
• R-groups of the amino acids of the active site at their optimal pH become ionized and allow ionic bonding between the substrate and the enzyme.
• At low pH, no active site/substrate binding will occur.
• The ionic bonding deformation makes enyzmes non functional/denatured.
• High amounts of hydroxyl ions effect R-group ionization & deform ionic bonding in basic environments.

Calculating Reaction Rates of Catalase and Hydrogen Peroxide

• To ascertain the initial reaction rate:
  • Determine the tangential slope of the curve.
  • Do so as close to time zero as possible.
• Plot the volume of oxygen collected over time.
• Graphically depict the beginning rates of reaction versus changes in substrate concentrations.
• There is a rate increase when an increased substrate concentration occurs, and the enzymes are occupied by the substrates.
• After, there is no further increase in the reactions rate, but substrate levels continues to increase,
• An enzyme is saturated when the active sites are fully substrate-occupied.
• Saturated enzymes operate at max velocity levels.
• Enzymes possess specific substrate affinities.
• Lack of enzyme-substrate affinity will not increase reaction, even after infinite concentration.

Michaelis-Menten Constant (Km)

• It is used to compare the affinity of different enzymes for their substrates.
• Km refers to the substrate concentration at which an enzyme works at half its maximum rate.
• Enzymes are fully occupied at the maximum stage.
• There needs to be a lower substrate for a higher enzyme affinity.

Inhibitors

• Inhibitors are biomolecules that slow enzyme action.
• Inhibitors lower the rate of reaction.
• They compete with molecules to bind with enzymes
• These can be split into two types:
  • Competitive reversible
  • Non-competitive reversible

Competitive reversible inhibitors

• Substrates convert the enzymes into products.
• They can compete with substrates to bind with enzymes at the enzyme's active site.
• The concentration levels determine outcome, a higher substrate will win.
• A competitive reversible inhibitor will bind as the active site, which prevents a permanent function of an enzyme function.
• The rate of reactions also decreases with a presence of a competitive inhibitor.
• Non-competitive do not affect the active site.
• They can disrupt hydrogen bonds which creates molecule enzyme shapes.
• Since the enzymes 3-D shape is distorted there will no longer be space for binding.
• They drastically reduce reaction rates.

Inhibitors and their Affects

• They only reduce the rate of the reaction; the reaction does not fall to zero.
• Inhibition is lethal when poisonous compounds prevent critical enzymes like cytochrome oxidase from producing cellular respiration.
• An example includes potassium cyanide.
• Cyanide inhibits the critical enzyme and causes cellular respiration to stop and can lead to death of cells.
• Major organ cells shut down in the lungs and heart regions
• Enzyme inhibition by inhibitors are sometimes essential, and not always bad.

Feedback Inhibition

• Metabolic reactions must be controlled and balanced.
• End-product usage is often used to control metabolic reactions.
• A substrate will bind with enzyme 1 which converts through intermediate products and into end-products, known as non-competitive reversible inhibition.
• Action is slowed down when enzymes increase, causing this product to bind to an area of the enzyme itself.
• This stops the enzyme from binding to its substrates, and will only reattach to continue elsewhere, which allows the reformation of new states.
• The enzyme will rise again when the product levels fall, which continues the cycle and creation of the product.
• End-product inhibition controls the product between upper and lower limits
• Feedback mechanisms are thus regulated as non-competitive.
• Catalase-hydrogen peroxide reactions happen.
• Enzymes will not get use up from the reaction.
• Remained will be in the remained even after the creation of new state.
• Enzymes on a solution will be lost once it is discarded, unlike a solution that has remained stable.

Enzyme Immobilization

• Enzyme immobilisation is where it gives advantages such as:
  • re-usage of reaction
  • yielding end-product is free from contamination
• The molecules are tolerant to changes in temperature/pH/ and held by alginate beads.
• The parts embedded in beads are the molecules often don't get exposed to change.
• When a mixture of sodium alginate and the enzyme are passed through calcium chordite, the beads will form.
• The immobilised enzyme functions at wider range of pH conditions.
• It does function a little lower in a state.
• Those enzymes speed up reactions and the molecules controlled.