Fig. 3.

2ʹ-d3′-AMP stimulates complementation in SYNAC without stimulating activity. (A) Effects of either 100 μM 2ʹ-d3′-AMP, 100 μM pyrophosphate (PPi), or both together (as indicated) on SYNAC activity in the presence of 100 μM FSK. Pyrophosphate is being generated from FSK-stimulated SYNAC activity and inhibits cyclase activity in conjunction with 2ʹ-d3′-AMP. In this assay, pyrophosphatase was added after the reaction had been stopped by denaturation at 65°C to remove pyrophosphate from solution, allowing detection by luciferase (see Materials and Methods). (B) The effects of 2ʹ-d3′-AMP, ATP, and pyrophosphate on SYNAC FRET in the presence and absence of 100 μM FSK. Counterintuitively, the FRET detected in the presence of both 2ʹ-d3′-AMP and pyrophosphate increases. Activity data are mean ± S.E.M. of three independent repeats using three different preparations of protein. FRET data are mean ± S.D. of three spectra. Significance assessed by Student’s t test (unpaired). n.s. = not significant; *P < 0.05; **P < 0.01.