Our investigation was sparked by the serendipitous observation that phosphorolysis reactions with 5bromouridine (1a) consistently exhibited noticeably lower equilibrium conversions in the presence of moderate concentrations of borate (Figures and). For instance, a reaction containing 400 µM 1a, 4 mM (10 equivalents) phosphate and 40 µg mL -1 (0.45 mol%) of the well characterized pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius (GtPyNP), serving as a model enzyme (see the Supplementary Information for details), reached its equilibrium at 70% conversion after 10 min in glycine-buffered solution (Fig. ). In contrast, the same reaction additionally containing 20 mM borate took almost 20 min to reach an equilibrium at 47% conversion, as monitored by multi-wavelength UV spectroscopy employing principles of spectral unmixing. This effect was not rooted in enzyme inactivation, as GtPyNP showed an identical melting point and fully retained its catalytic activity after prolonged incubation in borate-containing buffers (Figs. and). The observed apparent equilibrium shift persisted in the reverse direction of the reaction (starting from the products Rib1P and 2a, Fig. ) and additional experiments established that the reaction system consistently behaved according to lower apparent equilibrium constants of phosphorolysis (K′), whose magnitude depended on the concentration of borate (Fig. ). Since such drastic equilibrium shifts are unprecedented for phosphorolysis systems, we suspected that the formation of a dominant secondary species would actively remove the nucleoside 1a from this equilibrium in a thermodynamically controlled fashion. Indeed, the conversion data phenotypically following a Boltzmann-type relationship (Fig. ) could be described See the SI for experimental details, raw data and equations. [a] Not confidently accessible by DFT due to their high charge.