Among the substrates they studied were: ADP-ribose, CoA, ApppA, GDP, GTP, ITP, m\({}^{7}\)GpppG, NAD\({}^{+}\). Surprisingly, none of the compounds listed were classified as substrate for hNudt16 but instead were identified as specific for other members of the NUDIX hydrolase family, specifically ADP-ribose for Nudt5/Nudt9/Nudt12/Nudt14, ApppA for Nudt12/Nudt14, GDP for Nudt18, GTP for Nudt15, ITP for Nudt15, NAD\({}^{+}\) for Nudt12. No specific enzymatic activity against CoA and m\({}^{7}\)GpppG was detected among the proteins studied. Moreover, the authors did not propose any clear substrate for Nudt16. Biochemical and biophysical studies on different classes of substrates for hNudt16 (purine containing nucleotides, dinucleotides bearing triphosphate bridge, and short ribo-oligonucleotides) have identified the main determinants for substrate recognition [10; 12; 16; 17; 20]. Structural data for hNudt16 with ligands are available for IMP (PDB:2 XSQ), ADP-ribose (PDB:5W6X), diADDR (PDB:6B09), and FAD (PDB:6X7U). All these ligands are located inside the main binding pocket of hNudt16 in the vicinity of the metal ions. Several key residues that are crucial for hNudt16 activity are also located there [5; 10; 25]. Mutations R75L or E79Q have been found to reduce the enzymatic efficiency of the protein, and single mutations E76Q and E80Q, or the cluster double mutation E79K and E80K (EE-KK), can cause a complete loss of hydrolytic activity [10; 13]. However, the effects of these mutations on ligand-binding properties have yet to be studied. Given the inconsistent data on hNudt16 activity, we decided to investigate the substrate specificity of this protein. Our aim was to compare the specificity of hNudt16 towards different published substrates under the same experimental conditions to obtain quantitative data. For some published substrates, quantitative data are lacking, making very difficult to assess the significance of enzyme activity against this compound. It is necessary to systematize the data with a quantitative assessment of hNudt16 activity in order to further determine the biological role of this enzyme. We also decided to test hNudt16 activity against several new compounds that we found interesting. Here, we evaluated hNudt16 hydrolysis towards dinucleotide-containing diphosphates. Dinucleoside polyphosphate (XpnX; X = adenosine, guanosine, inosine or uridine, \(n\) = 2-7) have been shown to acts as extracellular mediators controlling numerous physiological functions, e.g., vascular tone or cell proliferation [26]. Two representatives of this group, GppG and AppA, were identified in human platelets and adrenal glands [26; 27]. Dinucleoside polyphosphate, mononucleosides, and mononucleoside polyphosphates together with enzymes and their corresponding receptors form a purinergic signaling system [28]. Guanidine-based purines (GTP, GDP, GMP, GUO, GUA) as a part of this system, have been shown to modulate intracellular processes such as growth, differentiation and survival [29]. It is therefore important to study the activity of hNudt16 against dinucleoside diphosphate, as this enzyme may be involved in controlling the levels of these compound, which affect important cellular processes. Our data showed a very fast catalytic process rate for the GppG compound. To have a more complete insight into substrate selection we decided to study not only the hydrolysis process but also the binding affinity of hNudt16 to broader list of selected substrates.