Currently I am testing a small collection of NRPS A-domains and I observed a relatively high activity in the absence of the artificial acceptor CoA. I know that similar observations have been made for certain CoA ligases, pointing towards release of the adenylate intermediate from the active site. Moreover, the activity of my A domains decreases in the presence of increasing amounts of CoA, which is consistent with their ping pong mechanism.

Furthermore, I was unable to detect the CoA adduct by LC/MS when reactions were performed in the absence of CoA. In contrast, this compound was readily observed when transformations were carried out in the presence of CoA. For activity measurements I am using a commercial pyrophosphate detection kit. What is your opinion about this?