In this context we became interested in the ratetemperature relationships of thermostable nucleoside phosphorylases (NPs) as these enzymes allow an experimental coverage of both a broad pH and temperature window. NPs perform the reversible phosphorolysis of nucleosides (Figure ) and are key catabolic enzymes in all kingdoms of life. Owing to their important role in various diseases, their catalytic properties have received considerable attention. Furthermore, NPs are used in synthetic chemistry for the preparation of pentose-1phosphates and nucleoside analogs and have been employed as analytical tools. While purine NPs have been researched intensively, including their ratetemperature relationships, comparably little is known about rate-temperature relationships of reactions catalyzed by the structurally distinct pyrimidine NPs. Since these enzymes typically operate across a wide pH window with similar rate constants and convert electronically diverse substrates, we hypothesized that they would present a convenient model system to interrogate pH and deprotonation effects on the Eyring plots of nucleoside phosphorolysis, as an example of a simple nucleophilic substitution. Thus, we questioned if there exists an activation heat capacity change during the phosphorolysis reactions catalyzed by thermostable pyrimidine NPs and, if so, whether this effect shows any pH-and/or protonationdependence across the broad working space of these enzymes. Herein, we report a kinetic characterization of the thermophilic pyrimidine NP from Geobacillus thermoglucosidasius (GtPyNP) across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values (thymidine, 1a, pKa ≈ 10.0 and 2'-deoxy-5-fluorouridine, 1b, pKa ≈ 7.3, Figure ).