Enzymes

• Enzymes are biological catalysts that speed up the rate of chemical reactions without being used up or changed
• Enzymes are also globular proteins with critical active sites where substrates bind
• Metabolic pathways are controlled by enzymes in a biochemical cascade of reactions
• Virtually every metabolic reaction within living organisms is catalyzed by an enzyme, making them essential for life to exist
• Intracellular enzymes are produced and function inside the cell, while extracellular enzymes are secreted by cells and catalyze reactions outside cells
• Enzymes work by lowering the activation energy of a reaction, influencing the stability of bonds in the reactants
• Enzymes and substrates are highly specific to each other, known as enzyme-substrate specificity
• Enzymes have an active site where specific substrates bind forming an enzyme-substrate complex
• The specificity of an enzyme is due to the complementary nature between the shape of the active site on the enzyme and its substrate(s)
• Enzyme reactions can be catabolic (breaking down molecules) or anabolic (building more complex molecules)
• The lock-and-key hypothesis describes enzymes and substrates as rigid structures that lock into each other precisely
• The induced-fit hypothesis explains that enzymes and substrates interact with each other, with conformational changes ensuring an ideal binding arrangement
• Enzyme activity can be investigated by measuring the rate of formation of a product or the rate of disappearance of a substrate
• Experimental set-up for investigating the rate of disappearance of a substrate using amylase:

  • Amylase and starch are combined and the reaction mixture is tested for starch at regular time intervals
  • Samples are taken from the reaction mixture at each time interval and added to iodine in potassium iodide solution
  • Starch forms a blue-black color with this solution, while the iodine solution remains yellow-brown if no starch is present
  • The time taken for starch to be broken down can be measured
  • The investigation can be repeated under different conditions by altering pH, temperature, enzyme concentration, or starch concentration, and then comparing the reaction rates
• Investigating the effect of starch concentration on amylase activity using colorimetry:

  • A colorimeter measures light absorbance or light transmission of a substance
  • Colorimetry can be used in any enzyme-catalyzed reaction that involves a color change
  • A calibration graph is plotted of starch concentration vs percentage absorbance or percentage transmission
  • Serial dilutions of starch are made to create a range of concentrations for the experiment
• Maths Skill: Drawing a Graph for Enzyme Rate Experiments:

  • Factors that can be changed in enzyme rate experiments include temperature, pH, enzyme concentration, and substrate concentration
  • Line graphs should be used to present the results, with the independent variable on the x-axis and the dependent variable on the y-axis
  • A line of best fit should be added to the graph to identify trends, and can be used for interpolation and extrapolation
• Maths Skill: Using a Tangent to Find Initial Rate of Reaction:

  • For non-linear enzyme rate experiment graphs, a tangent can be used to find the reaction rate at a specific point
  • The initial rate of reaction is the rate at the start of the reaction (where time = 0)
  • The gradient of the tangent drawn gives the initial rate of reaction
• Limiting Factors Affecting Enzymes: Temperature:

  • Enzymes have a specific optimum temperature where they catalyze a reaction at the maximum rate
  • Lower temperatures can prevent reactions or slow them down due to slower molecular movement and lower frequency of successful collisions between substrate molecules and active sites
• Enzymes have a specific optimum temperature where they catalyze a reaction at the maximum rate
• Lower temperatures slow down reactions due to molecules moving slower, resulting in lower frequency of successful collisions between substrate molecules and enzyme active sites
• Higher temperatures speed up reactions as molecules move faster, increasing the frequency of successful collisions between substrate molecules and enzyme active sites
• However, at very high temperatures, enzymes denature as bonds holding the enzyme molecule in its precise shape start to break, causing a sharp drop in the rate of catalysis
• Very few human enzymes can function at temperatures above 50°C, as temperatures exceeding 40°C can cause denaturation
• Enzymes have an optimum pH at which they operate best and are denatured at extremes of pH
• Solutions with excess H+ ions (acidic) or OH- ions (alkaline) can break hydrogen and ionic bonds in enzymes, altering the shape of the active site
• Enzyme concentration affects the rate of reaction, with higher concentrations increasing the likelihood of enzyme-substrate complex formation
• Substrate concentration also affects the rate of reaction, with higher substrate concentrations leading to a higher rate until all active sites become saturated
• Enzyme inhibitors can reduce or stop enzyme activity temporarily, with competitive inhibitors competing with substrates for the active site and non-competitive inhibitors binding to an alternative site, altering the shape of the active site
• Reversible inhibitors can act as regulators in metabolic pathways
• Metabolic reactions must be tightly controlled and balanced to prevent uncontrollable generation of a particular product
• End-product inhibition is a process where the end-product of a metabolic reaction chain binds to an alternative site on the enzyme, changing the shape of the active site and preventing the formation of further enzyme-substrate complexes
• Competitive inhibitors have a similar shape to substrate molecules and compete for the active site, while non-competitive inhibitors bind to an alternative site on the enzyme, altering the shape of the active site
• Increasing the concentration of an inhibitor reduces the rate of reaction; for competitive inhibitors, increasing substrate concentration can increase the rate of reaction, but for non-competitive inhibitors, the rate remains unchanged
• Enzymes have a wide variety of functions within organisms, catalyzing both intracellular and extracellular reactions that determine the structures and functions of cells and organisms
• In enzyme rate experiments, factors like temperature, pH, enzyme concentration, and substrate concentration can be changed, with only one variable changed per experiment (independent variable), while all other variables must be controlled (control variables)
• Uncertainty in enzyme rate experiments refers to the amount of error in measurements, with a small degree of uncertainty always present due to limited apparatus sensitivity
• Percentage error in enzyme rate experiments can be calculated using the formula: percentage error = (uncertainty ÷ measured value) x 100