pyruvate kinase calc

• Pyruvate


  A key crossroad in metabolism
• Importance of Pyruvate


  • Central Intermediate: Connects glycolysis to various metabolic pathways, acting as a key junction in metabolism
  • Versatility: Can be converted to acetyl-CoA, lactate, oxaloacetate, and alanine
• 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
• Forms of Pyruvate Kinase

  • Closed Form: Less active state, prevalent when inhibitory molecules are bound
  • Open Form: Active state, induced by binding of activating regulatory molecules
• Allosteric Regulation of Pyruvate Kinase


  Allosteric regulators like Fructose-1,6-bisphosphate (activator), ATP (inhibitor), and L-Alanine (inhibitor) modulate the activity of pyruvate kinase
• NADH Coupled Reaction to Measure Pyruvate Kinase Activity

  PEP+ADP→PKPyruvate+ATPPEP+ADPPK​Pyruvate+ATPPyruvate+NADH+H+→LDHLactate+NAD+
• NADH Absorption


  NADH absorbs light at 340 nm with an extinction coefficient (ε) of 6220 M⁻¹cm⁻¹
• Data Analysis


  Collect absorbance readings at 340 nm, plot absorbance vs time, calculate amount of NADH consumed, and determine reaction rate
• Practical Applications


  • Measure the effect of allosteric regulators (ATP, F1,6BP, Alanine) on pyruvate kinase activity
• There is a 1:1 ratio between pyruvate production and NADH consumption