prelab seminar

• Pyruvate


  A critical metabolic intermediate at the junction of several key pathways
• Metabolic Pathways Involving Pyruvate

  1. Glycolysis: Converts glucose to pyruvate, yielding ATP and NADH

  2. Citric Acid Cycle: Pyruvate is converted to acetyl-CoA and enters the citric acid cycle for further oxidation, producing more ATP, NADH, and FADH₂

  3. Lactic Acid Fermentation: In anaerobic conditions, pyruvate is reduced to lactate, regenerating NAD⁺ for glycolysis to continue

  4. Amino Acid Synthesis: Pyruvate serves as a precursor for the synthesis of amino acids like alanine
• Pyruvate entry into mitochondria

  • Tightly regulated, committing it to either energy production (via the citric acid cycle) or fatty acid synthesis
• Pyruvate Kinase (PK)


  Enzyme that catalyzes the final step of glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate and generating ATP
• Pyruvate Kinase Reaction


  PEP+ADP→Pyruvate+ATP
• Pyruvate Kinase


  • Allosteric Regulation: Enzyme activity is modulated by molecules binding at sites other than the active site
  • Closed Form: Inactive or less active state
  • Open Form: Active state induced by binding of regulatory molecules
• Fructose-1,6-bisphosphate (F1,6BP)


  Activator of Pyruvate Kinase, enhances its activity and promotes glycolysis
• ATP


  Inhibitor of Pyruvate Kinase, decreases its activity and signals sufficient energy levels
• 1. Alanine


  Inhibitor of Pyruvate Kinase, signals that pyruvate is being diverted for amino acid synthesis
• Measuring PK Activity


  1. Coupled Reaction: Use a coupled reaction to measure PK activity indirectly by observing changes in NADH absorbance at 340 nm

  2. NADH Coupled Reaction: NADH+H++pyruvate→NAD++lactate
• NADH Extinction Coefficient


  6220 M⁻¹cm⁻¹, making it useful for measuring enzyme reactions spectrophotometrically
• Determining Allosteric Effects


  1. Standard Assay: Establish a baseline PK activity

  2. Effect of ATP and F1,6BP: Compare PK activity in the presence of ATP (inhibitor) and F1,6BP (activator) using standard assay conditions
• Data Analysis


  1. Graphical Representation: Plot absorbance at 340 nm against time to visualize reaction progress

  2. Rate Calculation: Determine the rate of reaction (ΔA340nm/min) and convert to enzyme activity using Beer-Lambert's law

  3. Enzyme Activity: Expressed as µmoles of substrate processed per minute per mL of enzyme
• Visual Protocol for PK Assay

  1. Preparation: Set Up Reactions, Include PEP, ADP, NADH, lactate dehydrogenase, and the allosteric regulators ATP and F1,6BP

  2. Initiation: Add PK, Record the exact time of enzyme addition

  3. Measurement: Spectrophotometry, Collect absorbance readings to track the decrease in NADH concentration

  4. Data Analysis: Collect and record data points for absorbance over time, Plot absorbance versus time to determine reaction rates, Use the extinction coefficient of NADH to calculate enzyme activity in terms of µmoles of substrate processed per minute per mL of enzyme
• Importance of a Standard Reaction

  • Consistency: Ensures reliable and reproducible measurements.
  • Comparison: Allows for accurate comparison of enzyme activity under different conditions.
  • Validation: Confirms the effects of allosteric regulators on PK activity.
• Conclusion

  • Pyruvate Kinase Regulation: PK is a crucial regulatory point in glycolysis, controlled by allosteric effectors like F1,6BP and ATP.
  • Metabolic Integration: PK regulation links energy production with the cell's metabolic state and demands.
  • Experimental Skills: Gain hands-on experience in measuring enzyme activity and understanding the principles of allosteric regulation.