β asp lys

An Asp release assay (36 (link)) was used to detect Asp release from β-Asp-X dipeptides: Each 100 µL reaction contained 100 mM HEPES pH 8.2, 20 mM KCl, 5 mM α-ketoglutarate, 500 nM purified enzyme, 0.3 U malate dehydrogenase, 1 mM NADH, 2.4 U aspartate aminotransferase, and 1 mM dipeptide substrate. For other Arg-containing substrates, Arg release was monitored by using a free Arg detection kit (K-LARGE, Neogen, USA): The 135 µL reactions contained 15 µL buffer solution, 10 µL NADPH solution, 1 µL GIDH suspension, 2.5 µL urease solution, 1 µL arginase suspension, 1 mM substrate, and 500 nM enzyme. For both assays, reaction progression was monitored by following 340 nm transmittance in 96-well plates. Data were collected using a SpectraMax Paradigm (Molecular Devices, USA) and analyzed using GraphPad Prism (GraphPad, USA). β-Asp-Arg dipeptides were purified as previously described (26 (link)). β-Asp-Ala and α-Asp-Arg were purchased from Bachem (Switzerland), N²-acetyl arginine, β-Asp-Lys, and β-Asp-Leu from Toronto Research Chemicals (Canada), β-Asp-Asp from AchemBlock (USA), and N²-succinyl arginine from BLD Pharmatech (USA).