Enzyme assays

The initial rate of ATP cleavage was measured at varying concentrations of urea (0–50 mM) using a standard coupled ATPase assay. Assays were performed in 50 mM HEPES (pH 7.3), 50 mM NaCl, 8 mM MgSO₄, 8 mM NaHCO₃, 0‐50 mM Urea, 1.5 mM phosphoenolpyruvate, 0.15 mM NADH, 100 µM ATP, 5 U mL⁻¹ of pyruvate kinase and 12.5 U mL⁻¹ of lactate dehydrogenase. The reaction was initiated by the addition of enzyme (UC or UAL).

Allophanate hydrolase activity was assayed using a glutamate dehydrogenase coupled assay as previously described.49 The assay was performed in 50 mM HEPES (pH 7.3) and 50 mM NaCl, in the presence of 0.1–10 mM allophanate, 20 mM 2‐oxoglutarate, 0.15 mM NADH, and 20 U mL⁻¹ glutamate dehydrogenase. Reactions were initiated by the addition of AH (∼2.5 μg mL⁻¹).

The UC‐AH coupling activity and substrate channeling activity of UC and AH was assayed using the glutamate dehydrogenase coupled assay in a buffer containing 50 mM HEPES (pH 7.3) and 50 mM NaCl and in the presence of 0.15 mM NADH, 8 mM MgSO₄, 50 mM Urea, 100 µM ATP, 8 mM NaHCO₃, 10 mM 2‐oxoglutarate and 20 U mL⁻¹ of glutamate dehydrogenase. For the substrate channeling assays, the overall activity of UC and AH was measured as described above, with increasing concentrations of PsAH_S179A titrated into a 1:1 molar ratio mixture of UC (90 μg mL⁻¹) and AH (45 μg mL⁻¹). To simulate a crowded cellular environment, 22% (w/v) PEG4000 was added to the assay buffer.