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. Author manuscript; available in PMC: 2020 Dec 18.
Published in final edited form as: J Am Chem Soc. 2019 Dec 9;141(50):19625–19633. doi: 10.1021/jacs.9b07111

Figure 2 ∣. 18O incorporation into phosphate in multi-turnover reaction.

Figure 2 ∣

(A) ESI-MS analysis of GluC digested 18O labeled TglA. (B) 31P NMR analysis shows 18O incorporation into phosphate when using 50 μM 18O labeled TglA for a multiple turnover reaction catalyzed by 5 μM TglB in the presence of 50 μM Cys-tRNACys, 0.5 mM ATP, and 5 mM MgCl2. (C) Two possible mechanisms that could explain the 18O incorporation into phosphate in a multi-turnover reaction. In mechanism I, ATP-dependent phosphorylation occurs on a carboxylic acid residue on TglB, and subsequently involves the intermediacy of a TglA-TglB anhydride. This would result in 18O-labeled TglB, which could deposit the label into phosphate in subsequent turnovers. In mechanism II, it is the C-terminal carboxylate of TglA that is directly activated during reaction.