Collectively, these results provide several insights. First, despite disagreeing literature, this untangling of activity and denaturation effects revealed that GtPyNP possesses no classical optimum temperature. Previous assignments of an optimum temperature of this enzyme can likely be ascribed to enzyme denaturation since its theoretical optimum temperature is approximately equal to its melting temperature (TM = 77.5 °C, theoretical Topt = 75 °C for 1a or 79 °C for 1b, Figures and). Secondly, the data presented above lend further evidence to the notion that binding of the substrate and binding of the transition state are unrelated phenomena, as enzymes primarily discriminate between substrates on the transition state level rather than via selective binding of the ground states of substrates. This is evident here in the observed difference of the affinities for the two substrates (KM = 0.6 mM for 1a and 3.1 mM for 1b) and the similarity of Δ ‡ (-1.9 and -1.2 kJ mol -1 K -1 ). Thirdly, considering previous work indicating that the overall phosphorolysis of 1a and 1b proceeds with almost the same equilibrium state thermodynamics, these results indicate that electron withdrawal by the fluorine substituent primarily has a kinetic effect in this reaction system and almost no thermodynamic one. This is reflected by the lower Δ ‡ for 1b compared to 1a contrasting their almost identical equilibrium constants (Keq = 0.15 for 1a and 0.12 for 1b) and apparent net thermodynamic parameters Δ and Δ (Scheme S2). Next, we extended this kinetic characterization to the entire pH range accessible with GtPyNP to examine the impact of reaction pH and substrate deprotonation on the kinetic behavior of this system. We hypothesized that the medium pH might influence the surface charge of the protein and, consequently, its molecular dynamics, which might be reflected by the heat capacity changes during catalysis. Furthermore, 1b (pKa ≈ 7.3) becomes fully deprotonated at higher pH values and we expected to see pH effects on its activation thermodynamics. To probe these hypotheses, we obtained Eyring plots for the phosphorolysis of 1a and 1b from pH 7-10 in steps of 0.5 pH units (Figure ). In all but one condition, the AIC supported the MMRT model ( ) as the better fit, substantiating the presence of a negative Δ ‡ (Table , Figure ). Unexpectedly, the reaction pH generally had no significant impact on the phosphorolysis kinetics with 1a as and all fit parameters remained largely unchanged across the working space (Figure ). Despite its deprotonation in the upper portion of the working space, a similar situation existed for 1b, albeit with a much lower and error-prone Δ ‡ (Figure ). Although deprotonation severely affected GtPyNP's affinity for this substrate (KM >10 mM at pH 9 compared to 3.1 mM at pH 7, Figure ), it had no significant effect on the thermodynamic activation parameters, suggesting that transition state formation and binding are unaffected by the net charge of this substrate. Most likely, deprotonation of the substrate occurs per se in the active site of the enzyme where an anionic species would be well stabilized by positively charged residues (Scheme S1 and e.g. pdb ID 1uou or 2wk6). As such, considering this mode of substrate complexation in pyrimidine NP active sites, it is reasonable to assume that even a partial dianion present during such a transformation can be accommodated (Scheme S1). Overall, the results of these experiments indicate that GtPyNPcatalyzed phosphorolysis is strikingly robust and shows little sensitivity to pH shifts or the protonation state of its nucleoside substrate, despite charge delocalization being a primary mechanism of rate acceleration in pyrimidine NPs. The fact that we obtained very similar Eyring plots over a broad pH range further indicates that the surface charge of the protein insignificantly contributed to the molecular dynamics and, in extension, activation heat capacity changes along the reaction coordinate. Nonetheless, it remains to be demonstrated if this behavior observed for the dimeric GtPyNP with a solvent-shielded active site also translates to highly multimeric enzymes or those with solvent-exposed active sites.