Enzchek pyrophosphate assay

The specific activity of the T336I and F361V ASD-linked ASNS variants was determined using the EnzChek^TM Pyrophosphate Assay (Molecular Probes). Briefly, reaction was initiated by addition of 0.1 μM enzyme to a reaction mixture containing 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 10 mM l-glutamine, 10 mM l-aspartate, 5 mM ATP, 0.2 mM MESG, 1 U mL⁻¹ purine nucleoside phosphorylase, and 0.03 U mL⁻¹ inorganic pyrophosphatase at 37 °C in 100 mM EPPS buffer, pH 8.0. The activity was monitored by measuring the absorption change at 360 nm using a Varian Cary® 50 UV-visible spectrometer (Agilent Technologies). A standard curve was determined using standard MgPP_i solution diluted in a buffer solution of 100 mM EPPS, pH 8.0, containing 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 10 mM l-glutamine, 10 mM l-aspartate, 5 mM ATP, 0.2 mM MESG, 1 U mL⁻¹ purine nucleoside phosphorylase, and 0.03 U mL⁻¹ inorganic pyrophosphatase at 37 °C. The percentage activity of each variant was normalized against that of WT human ASNS (Supplementary Fig. 10).

The activity of WT human ASNS and four ASNS variants (E364A, E364Q, D367A, and D367N) was assayed by measuring the rate of MgPP_i production using a continuous assay employing the EnzChek^TM Pyrophosphate Assay (Molecular Probes) as described in detail elsewhere^{96 (link)}. In these experiments, assay mixtures contained 0.5 mM ATP, 5 mM l-aspartate 0.01 U mL⁻¹ nucleoside phosphorylase, 3 × 10⁻⁴ U mL⁻¹ inorganic phosphatase, and 100 mM NH₄Cl dissolved in 100 mM EPPS buffer, pH 8.0, containing 10 mM MgCl₂ and either 0 μM or 1 μM ASNS inhibitor 1 (as a 1:1 mixture of epimers 1a and 1b) (1 mL total volume). Reactions were initiated at 25 °C by the addition of recombinant, WT human ASNS (4 μg), and 2-amino-6-mercapto-7-methylpurine production was then monitored spectrophotometrically at 360 nm over a period of 10 min. All kinetic assays were performed in triplicate. Identical assay conditions were employed in studies of the D367N ASNS variant except that l-aspartate was present at a final concentration of 50 mM and reactions were initiated by the addition of recombinant enzyme (40 μg). A standard curve to convert absorbance units into MgPP_i concentration was constructed using known amounts of MgPP_i dissolved in 50 mM EPPS buffer, pH 8.0, containing 50 mM L-aspartate, 200 mM NaCl and 2 mM TCEP. The standard curve was unaffected by the presence of up to 10 μM ASNS inhibitor 1.

Diguanylate cyclase activity of purified recombinant TdcA (His₆-MBP-TdcA or His₆-NusA-TdcA) and recombinant WspR (His₆-WspR) was measured using an EnzChek® Pyrophosphate Assay (Invitrogen) with modifications⁷⁰ also summarized here. High-purity guanosine 5ʹ-triphosphate (GTP) was purchased from Affymetrix. Yeast pyrophophatase was purchased from Roche. A standard curve was produced for each temperature using 50, 75, 100, and 125 μM pyrophosphate. We used 0.5 mM GTP for all measurements of diguanylate cyclase activity. Data were collected using a Lambda 35 UV/VIS Spectrometer with a Peltier device (Perkin-Elmer). Measurements were done using SUPRASIL® quartz spectroscopy cells with a light path of 10 mm (Perkin-Elmer).