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bioRxiv · 10.1101/2024.06.27.601032

Free Amino Acids Accelerate the Time-Dependent Inactivation of Rat Liver Nucleotide Pyrophosphatase / Phosphodiesterase Enpp3 elicited by EDTA

Romero, A.·Cumplido-Laso, G.·Fernandez, A.·Moreno, J.·Canales, J.·Ferreira, R.·Lopez-Gomez, J.·Ribeiro, J. M.·Costas, M. J.·Cameselle, J. C.
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Abstract

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2,3-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2,3-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2,3-cGAMP, and that Enpp1 and Enpp3 account for all the 2,3-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2,3-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

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Romero, A., Cumplido-Laso, G., Fernandez, A., Moreno, J., Canales, J., Ferreira, R., Lopez-Gomez, J., Ribeiro, J. M., Costas, M. J., Cameselle, J. C.. 2024-06-27. Free Amino Acids Accelerate the Time-Dependent Inactivation of Rat Liver Nucleotide Pyrophosphatase / Phosphodiesterase Enpp3 elicited by EDTA. https://doi.org/10.1101/2024.06.27.601032

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