Lecture 03

• What is the focus of the study material?

  Introduction to Enzyme Kinetics
• What should be considered when choosing substrate concentration ranges?

  • Best substrate concentration range
  • Lowest and highest [S]
  • Five evenly spread intermediate concentrations
  • Cover at least 90% of saturation curve
• What is the substrate range?

  Difference between lowest and highest concentrations of [S]
• How many intermediate concentrations should be determined?

  Five intermediate concentrations
• What is the purpose of using a range of concentrations?

  To cover at least 90% of the saturation curve
• How is the % saturation of the enzyme calculated?

  Using the modified Michaelis-Menton equation
• What does Vmax represent in enzyme kinetics?

  Velocity when all enzyme molecules have substrate bound
• What does Vo represent in enzyme kinetics?

  Velocity when less than 100% of enzyme molecules are bound
• What is the formula for % saturation of the enzyme?

  % saturation = Vo/Vmax
• How is the % saturation of enzyme with substrate expressed?

  % saturation = [S] / (Km + [S])
• What happens when substrate concentration equals Km?

  Reaction velocity is 50% of Vmax
• What is the reaction velocity when substrate concentration is 0.3x Km?

  23.1% of Vmax
• What is the reaction velocity when substrate concentration is 3x Km?

  75% of Vmax
• What are the applications of Vmax and Km?

  • Determining a natural substrate for an enzyme
  • Assessing catalytic efficiency
  • Evaluating specificity for a substrate
• How is catalytic efficiency calculated for hexokinase?

  Using the ratio kcat/Km or Vmax/Km
• What factors are considered for hexokinase's physiological substrate?

  • Physiological concentration of sugar in the brain
  • % of hexokinase with sugar bound
  • Vmax/Km ratio for glucose and fructose
• What is the % saturation of hexokinase with glucose?

  50% saturation
• How is the % saturation of hexokinase calculated with glucose?

  % Saturation = [S]/([S] + Km)
• What is the % saturation of hexokinase with fructose?

  0.09% saturation
• How is the % saturation of hexokinase calculated with fructose?

  % Saturation = [S]/([S] + Km)
• What are the percentages of hexokinase bound to glucose and fructose?

  • Percentage of hexokinase bound to glucose = 50%
  • Percentage of hexokinase bound to fructose = 0.1%
• What is the saturation of hexokinase under physiological conditions with glucose?

  50% saturation
• What is the saturation of hexokinase under physiological conditions with fructose?

  0.09% saturation
• How much more glucose is bound to hexokinase than fructose?

  556 times more glucose
• What are the key points regarding hexokinase mutations and type 2 diabetes?

  • How to calculate initial velocities
  • Obtaining Km and Vmax values using graphs
  • Determining natural substrate using Vmax/Km
  • Degree of saturation at specific substrate concentration