PycA Protein Catalytic Activity Assay

The wild-type PycA protein was expressed and purified as previously described (Sureka et al., 2014 (link)). pycA mutations were made using the QuikChange kit (Stratagene) and confirmed by Sanger DNA sequencing. The mutant alleles were expressed and purified using the same protocol as the wild-type protein. The catalytic activities of wild-type and mutant proteins were assessed based on a published protocol (Modak and Kelly, 1995 (link)), which couples oxaloacetate production to the oxidation of NADH by malate dehydrogenase (Sigma), followed spectrophotometrically by the decrease in absorbance at 340 nm. The activity was measured at room temperature in a reaction mixture containing 100 mM Tris (pH 7.5), 200 mM NaCl, 5 mM MgCl₂, 50 mM NaHCO₃, 50 mM (NH₄)₂SO₄, 5 units of malate dehydrogenase, 4 mM NADH, 5 mM pyruvate, 2 mM ATP, and 1 μM PycA (based on the monomer). The reaction was initiated by addition of ATP.