Pyruvate kinase enzyme assay and kinetics

The activity of PK was measured as the rate of production of pyruvate from PEP in a coupled enzyme reaction with lactate dehydrogenase that measured the rate of NADH consumption with bovine heart lactate dehydrogenase (CAS 9001-60-9, Sigma-Aldrich). This was performed by measuring the absorbance at 340 nm using a BioTek spectrophotometer on a 96-well microplate. The optimal assay conditions for red muscle PK in the forward PEP consuming direction were 37.5 mM Tris pH 7.2, 7.45 mM KCl, 0.15 mM NADH, 0.5 mM PEP, 0.5 mM ADP, 5.5 mM Mg⁺⁺, 1 U of LDH and 10 µL of enriched PK enzyme in a 200 µL reaction volume. The optimal conditions for liver PK in the forward direction were 36.25 mM Tris pH 7.2, 7.25 mM KCl, 0.15 mM NADH, 1 mM PEP, 1 mM ADP, 5 mM Mg⁺⁺, 1 U of LDH, and 10 µL of enriched enzyme. Assays were typically initiated through the addition of PEP. As PK has an extremely high and negative ΔG only the activity in the forward direction was measured. All assays were run at room temperature (22 °C). K_m values for PEP and ADP and I₅₀ values for KCl and urea were determined at constant, saturating co-substrate concentrations, as above. To determine the effect of pH on the K_m of PEP, all assay concentrations were the same as above, except 36.25 mM potassium phosphate at pH 6.6 was substituted for Tris. I₅₀ values for ATP, L-alanine, and lactate and the K_a values for FBP and aspartate were determined at constant, saturating levels of substrate. Kinetic data were analyzed using the Kinetics v.3.5.1 program (Brooks, 1992).