Lecture 01, 2

• What is the focus of Enzyme Kinetics?

  Determining kinetic parameters in enzymology
• How do enzymes change in pathological states?

  They alter their concentrations in diseases
• How can kinetic parameters help identify an enzyme's natural substrate?

  By using values from kinetic analysis

  
• What does the initial rate refer to in enzyme kinetics?

  Rate of product formation over time
• What do Km, Vo, Vmax, and kcat represent?

  Kinetic parameters in enzyme reactions
• How can you graphically calculate Km, Vo, and Vmax?

  By plotting product concentration vs. time

  
• What issues can lead to non-linear initial rate kinetics?

  Problems in assay conditions or enzyme dilution
• What is a limitation of initial rate kinetics?

  It may not reflect true enzyme activity
• How can kinetics be applied in drug design?

  By understanding in-cell metabolism
• What is the role of ACE in the body?

  It is involved in hypertension
• What does the gastric H+ ATPase do?

  It secretes gastric acid and aids ulcers
• What is the function of liver cytochrome P450?

  It metabolises toxins and pharmaceuticals
• What do ABC ATPases contribute to?

  Resistance to antimicrobials and anti-cancer drugs
• What does the term "steady state" refer to in enzyme kinetics?

  Constant concentration of enzyme-substrate complex

  
• How is the initial rate obtained from a reaction progress curve?

  By taking the tangent at the curve's start
• What is kcat also known as?

  Turnover number or catalytic constant
• How is Vmax related to kcat?

  Vmax = kcat x [Etrue]
• What is the difference between true enzyme concentration and total enzyme concentration?

  True concentration includes only active enzyme
• How does Vmax change with enzyme concentration?

  It increases if enzyme concentration is doubled
• What is Km in enzyme kinetics?

  Michaelis constant, a concentration term

  
• What does Vo = 0.5Vmax indicate?

  Half saturation of the enzyme
• How do you determine kinetic parameters like Km and Vmax?

  Using non-linear regression analysis
• What happens to Vo as substrate concentration increases?

  Vo increases until it reaches Vmax
• What is a saturation plot in enzyme kinetics?

  Graph of Vo vs. substrate concentration

  
• What does the Michaelis-Menten equation simplify to at low substrate concentrations?

  First order reaction with respect to [S]

  
• What factors can cause upward curvature in kinetics?

  Temperature control and inhibitor loss
• What can lead to downward curvature in kinetics?

  Enzyme inactivation over time
• What is the goal of measuring initial rates in enzyme kinetics?

  To create a saturation plot

  
• How do you decide the range of substrate concentrations for a reaction?

  By covering at least 90% of the saturation curve
• How do you calculate % saturation of an enzyme?

  Using Vo/Vmax = ([S])/(Km + [S])
• What does a Km value of 3 mM indicate in an example?

  It is the concentration for half-maximal velocity
• What range of substrate concentrations should be used for effective saturation coverage?

  0.3 Km to 3 Km or 0.1 Km to 10 Km
• How many points are needed to cover a saturation curve effectively?

  At least 7 points for accuracy
• What is the minimum number of points needed to plot a straight line?

  At least 2 points are required
• How many data points are needed if the curve is broken into four straight lines?

  9 points are needed for accuracy
• What is the significance of having points both above and below Km?

  It provides a comprehensive view of kinetics
• What is the purpose of the Lineweaver-Burk graph?

  To calculate Vmax and Km values
• How do you determine the substrate range for experiments?

  By identifying lowest and highest substrate concentrations
• How do you ensure a range of concentrations covers 90% of the saturation curve?

  By evenly spreading five intermediate concentrations
• What does the modified Michaelis-Menten equation represent?

  Relationship between Vo, Vmax, Km, and [S]