ATP-[32P]PPi exchange assay of full-length ClbH, ClbH-A1, ClbH-C-A2-PCP, and ClbH-C-A2-PCP S1524A (Supplementary Fig. 4)

Assays (100 μL) contained 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl₂, 5 mM dithiothreitol, 5 mM ATP, 1 mM amino acid, and 4 mM Na₄PPi/[³²P]PPi (2–6 × 10⁵ cpm/mL; Perkin Elmer). Reactions were initiated by the addition of enzyme (1 μM) and incubated at room temperature for 30 min. Reactions were quenched by the addition of 200 μL of charcoal suspension (16 g/L activated charcoal, 100 mM Na₄PPi, 3.5% [v/v] HClO₄). The samples were centrifuged (13,000 rpm × 3 min), and the supernatant was removed. The charcoal pellet was washed twice with 200 μL of wash buffer (100 mM Na₄PPi, 3.5% [v/v] HClO₄). The pellet was re-suspended in 300 μL of wash buffer and added to 10 mL of scintillation fluid (Ultima Gold, Perkin Elmer). Radioactivity was measured on a Beckman LS 6000SC scintillation counter.