represent approximate values at 25 °C and are subject to slight changes at higher temperatures. Data in B were acquired via sampling and discontinuous monitoring of reactions with a high-throughput assay and fitted to the Eyring (1) and MMRT (2) equations, as described in the Supplementary Information. Please see the externally hosted Supporting Information for raw data and calculations. First, we assayed GtPyNP for phosphorolytic conversion of 1a and 1b at pH 7, employing deconvolution of UV absorption spectra for reaction monitoring. To this end, several preliminary experiments were essential, as we aimed to acquire activity data unaffected by denaturation effects (TM = 77.5 °C, Figure ), which required the selection of experimental conditions enabling sufficient stability of the enzyme (please see the Supplementary Information for details). Employing saturating substrate concentrations and a temperature window of 30-65 °C, the methylated nucleoside 1a was converted with observed rate constants of 2.1-30.3 s -1 , showing a clear temperaturedependence (Figure ). Both the Eyring and the MMRT equation provided reasonably good fits of the experimental data (R 2 = 0.84 and 0.86). However, the Eyring fit showed a systematic deviation from the data by overestimating at the extremes of the temperature and underestimating in the middle of the dataset, while also suggesting a considerable enthalpy-entropy trade-off (Δ ‡ = 49.9 kJ mol -1 and Δ ‡ = -71.8 J mol -1 K -1 ). In contrast, the MMRT fit provided a more realistic description of the dataset by reflecting the experimental data more evenly throughout the full temperature range, as supported by the Akaike information criterion (AIC, Table ). This fit yielded a heat capacity change Δ ‡ of -1.9 ± 0.7 kJ mol -1 K -1 , manifesting itself in a moderate but significant curvature in the graph. A similar observation was made for the fluorinated nucleoside 1b, whose phosphorolysis showed a Δ ‡ of -1.2 ± 0.6 kJ mol -1 K -1 . This suggests a comparable binding of the transition states of both substrates, which is likely rooted in the nearly equal steric demand of these substrates as well as their identical electronic interactions with the H-bonding residues in the GtPyNP active site under these conditions. In contrast to 1a, the fluorinated 1b was converted with much higher rate constants of 69.7-406.6 s -1 . Interestingly, this increase of originated from a significantly lower activation enthalpy (Δ !"# ‡ = 92.2 kJ mol -1 for 1a and 62.5 kJ mol -1 for 1b), which outweighed the lower entropic contribution (Table ) and most likely reflects a weaker glycosidic bond caused by electron withdrawal through the fluorine substituent. In line with the lower activation enthalpy observed for 1b, its phosphorolysis showed a shallower temperaturedependence, reflected by a rate increase of factor 4.9 between 30 and 65 °C, compared to a factor of 8.6 for 1a.