An Optimized Enzyme-Coupled Spectrophotometric Method for Measuring Pyruvate Kinase Kinetics.

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, producing ATP in the final step of glycolysis. Unlike other isoforms, PKM2 is uniquely regulated, shifting between active tetramers and less active dimers to balance energy production with biosynthetic demands. This flexibility is exploited in cancer cells to support the Warburg effect and anabolic growth. Additionally, PKM2 can translocate to the nucleus and act as a transcriptional co-activator, influencing gene expression and tumor progression. To facilitate functional studies of PKM2, we present a robust and reproducible protocol for its expression, purification, and enzymatic characterization. PKM2 is expressed in and purified via Ni-NTA affinity and size-exclusion chromatography to ensure high purity and proper folding. Enzymatic activity is measured using a lactate dehydrogenase (LDH)-coupled assay that tracks NADH oxidation at 340 nm, allowing sensitive kinetic analysis under various conditions, including different PEP concentrations, pH levels, and presence of the allosteric activator fructose-1,6-bisphosphate (FBP). This non-radioactive, high-resolution method is suitable for analyzing PKM2 regulation, post-translational modifications, and mutant variants, as well as for screening potential therapeutic modulators, providing a valuable tool for cancer metabolism research. Key features • Enables robust and scalable expression of recombinant wild-type PKM2 in , yielding protein suitable for biochemical and structural studies. • Utilizes a non-radioactive, LDH-coupled spectrophotometric assay to accurately measure PKM2 enzymatic activity in real time by monitoring NADH consumption at 340 nm. • Supports kinetic analysis under physiologically relevant conditions, including variable pH and in the presence or absence of the allosteric activator fructose-1,6-bisphosphate (FBP). • Suitable for comparative activity profiling of PKM2 variants, mutants, or post-translationally modified forms.