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. 2017 May 9;12(5):e0177270. doi: 10.1371/journal.pone.0177270

Fig 3. An overview of dissociation constants measured in this study.

Fig 3

For those complexes that gave poor c-values in the ITC, Kd values were determined by NMR (marked by *). A summary of Kd values is presented in Table 1. (A) Determination of Kd values for the interaction of free bmAANAT3 with acetyl-CoA (black) and CoA (gray), and DAT with acetyl-CoA (red). The isotherms presented in the middle panel correspond to interaction of bmAANAT3 with acetyl-CoA and CoA (black) and to the interaction of DAT with acetyl-CoA (red). NMR was used to confirm the Kd of bmAANAT3 for CoA (right panel). (B) Determination of Kd values for the interaction of different cofactor-liganded states of bmAANAT3 with tryptamine and tryptamine analogs. The presence of acetyl-CoA in the catalytic funnel leads to an increase in the affinity for tryptophol, while an even more substantial increase is observed when an acetylated derivative of tryptamine (N-acetyltryptamine) is utilized. Binding of N-acetyltryptamine to bmAANAT3 is not sensitive to the presence of CoA, but it is abolished in the presence of acetyl-CoA. The isotherms presented in the middle panel correspond to the interaction of bmAANAT3 with N-acetyltryptamine in the free- or CoA-bound states (black) and in the acetyl-CoA-bound state (red). NMR was used to measure the Kd for the interaction of free- and acetyl-CoA-bound bmAANAT3 with tryptophol (right panel). (C) Determination of Kd values for the interaction of different cofactor-liganded states of bmAANAT3 and variants (D26A) with tyramine and tyramine analogs. The presence of CoA in the catalytic funnel leads to an increase in the affinity for tyrosol, while an even more substantial increase is observed in the presence of acetyl-CoA. The D26A mutation renders tyrosol binding insensitive to acetyl-group occupancy of the catalytic center, in a manner that tyrosol affinity of acetyl-CoA-bound bmAANAT3D26A is similar to that of CoA-bound bmAANAT3D26A or CoA-bound bmAANAT3. NMR was used to measure the Kd for the interaction of tyrosol with free- or CoA-bound bmAANAT3, and for acetyl-CoA- or CoA-bound bmAANAT3D26A.