## 3 Discussion  
According to previous reports, hNudt16 can hydrolyze the canonical cap of cytoplasmic mRNAs and small nucleolar RNAs, eliminate dIDP/IDP and dITP/ITP, and process ADP-ribosylation of proteins, DNA, and RNA, as well as non-canonical metabolites present on some cellular RNAs [5; 8; 9; 10; 11; 13; 14; 15; 17; 18; 19; 20; 21; 22; 23]. However, it is difficult to determine whether all of the activities listed are biologically relevant. Particularly that some of the published results contradict each other [24]. To gain a better understanding of the activity of hNudt16, the present study focused on a quantitative comparison of the specific properties of hNudt16 with previously published substrates, as well as other substrates that have not yet been investigated for this protein. In our study, we screened and compared 15 compounds: GppG, GpppG, m\({}^{7}\)GppG, m\({}^{7}\)GpppG, GMP, GDP, m\({}^{7}\)GDP, IMP, IDP, CDP, dpCoA, AppA, ADPr, NAD, and NADH. Both the wild-type protein and its mutants were investigated. Biophysical studies of different hNudt16 variants were performed to identify a variant that was able to bind the substrate but not hydrolyze, namely E76Q. The E76Q variant was the optimal replacement for the wild-type protein and was used as a model for a non-hydrolytically active protein. Our biophysical studies showed that the variant had an unchanged secondary structure and thermal stability. E76Q also showed the ability to bind magnesium ions, but most likely at only one position. The fact that the E76Q mutation did not affect ligand binding indicates that one magnesium ion coordinated by Glu80 [17] is sufficient for ligand binding. However, the mutation of any of the amino acids involved in the coordination of the second magnesium ion will impair the catalytic mechanism. Protein-ligand binding studies with hNudt16E76Q showed that this mutant has the highest binding affinity towards IDP and slightly lower, but of the same order of magnitude, for GppG. Furthermore, wild-type hNudt16 had very high hydrolysis rate in activity screening assays with GppG. The DSF and MST results obtained for mutant E76Q with the tested ligands were consistent with results obtained from enzymatic assays for wild-type protein, and lower hydrolysis parameters were directly related to weaker binding affinity. Ligands with diphosphate bonds have a few orders of magnitude higher affinity than mono- and tri-phosphates for identical nucleotides, making the diphosphate substrates the most favored by hNudt16. Thus, we can conclude that the number of phosphate residues was essential for hNudt16 binding affinity and hydrolytic efficiency. This is consistent with the structure of X29 (homolog of hNudt16 from _X. laevis_) with GTP, where terminal phosphate (P\(\gamma\)) is surrounded by acidic amino acids that may be involved in the formation of an acid patch that negatively affects the binding of triphosphate nucleotides in comparison to its diphosphate counterpart [17; 25]. Interestingly, ligands with unmethylated nucleotides are not only hydrolyzed by hNudt16 with greater efficiency, as reported previously by [PERSON] et al. [12] but they also have higher binding affinity and thermal stability than methylated ligands. Binding affinity and hydrolytic studies showed that ligands such as NAD, NADH, free ADP, or dpCoA are poor substrates for the hNudt16 enzyme (which was also shown in the case of radioactively labeled NAD [13], or ADPr with K\({}_{\text{M}}\sim 1\) mM [19]). hNudt16 has been reported to hydrolyze NAD and dpCoA moieties in vitro and in vivo when they are attached to 5'-RNA (metabolite caps) [13], or polyADPribosylated proteins [19]. This suggests that effective hydrolysis of these structures may depend on the interaction of hNudt16 with the remaining part of the processed molecule (e.g., RNA body). A potential positively charged channel for RNA binding was proposed based on the crystal structure of X29 [25].