Enzyme Kinetics of PKAL-1 via Coupled Assay

The enzyme kinetics of PKAL-1 were determined through an enzyme-coupled spectrophotometric assay^{35 (link)}. Each 100 μL assay mixture contained 100 mM Tris buffer pH 7.4, 1 mM dithiothreitol, 10 mM MgCl₂, 4 mM ATP, 0.9 mM phosphoenolpyruvate, 0.3 mM NADH, 2.5 U pyruvate kinase, 3.5 U lactate dehydrogenase, 10 U adenylate kinase (Sigma M3003, prepared according to manufacturer’s protocol), 100 mM buffered hydroxylamine, and the tested substrates in DMSO (final volume 2.5%). The kinetic assay was initiated by the addition of an enzyme and run at 22 °C. No activity was detected in the assay when initiating with PKAL-1(K488A). GraphPad Prism was used to calculate the apparent kinetic constants.