Next, we sought to identify the cause of the apparently reduced reaction rates in the presence of borate by enlisting kinetic studies and molecular dynamics (MD) simulations. This effect was especially prominent with borate concentrations greater than 20 mM and led to decreases in the reaction rate of GtPyNP by more than a factor of four, as illustrated in Figure . Since high borate concentrations primarily yield 1a* in solution (and not the enzymatic substrate 1a), we initially entertained the hypothesis that this phenomenon was a function of the decreased concentration of the free nucleoside substrate and the associated apparent decrease of affinity. However, the Michaelis-Menten kinetics obtained for 1a (with phosphate in excess) proved inconsistent with this hypothesis. While a reduction in available substrate concentration should primarily result in a decrease in the apparent Michaelis-Menten constant KM, we observed no change in KM but instead a sharp decline of the rate constant under saturating substrate concentrations (kcat, Fig. ). With saturating concentrations of both substrates, the observed rate constant kobs exhibited a similar Boltzmann-type decrease as observed for the apparent equilibrium constant, which could be described well by an equilibrium model expressing the observed rate constant as a function of kcat and the esterified fraction of 1a (Fig 2D, see the SI for equations). As this decrease of kobs was further completely absent for deoxy-1a in the presence of borate (Figs. and), we concluded that borate alone does not inhibit GtPyNP, but rather the borate ester 1a*. If GtPyNP could bind but not convert 1a*, we reasoned that the position of the equilibrium between 1a and 1a* would determine the ratio of potentially active enzyme (1a bound to GtPyNP) versus inactive enzyme (1a* bound to GtPyNP), assuming that catalysis is a rate-limiting step. Given the highly solvent-exposed active site of pyrimidine nucleoside phosphorylases in the open state, we expected that GtPyNP should be able to accommodate the slightly twisted and sterically more demanding borate ester 1a* and allow its equilibration with the free nucleoside substrate 1a while bound to the enzyme. Phenomenologically, such a process would resemble a classical non-competitive inhibition, consistent with our kinetic data. Indeed, MD simulations (using GROMACS with the CHARMM36 force field) based on our recently disclosed crystal structure of GtPyNP in complex with uridine (PDB ID 7m7k, see the SI for details) yielded several insights in support of the proposed model. First, an analysis of the clustered states over 50 ns simulation time indicates that the borate ester 1a* can be bound in analogy to 1a via hydrogen bonds with the amide motif of the nucleobase. Secondly, this analysis also showed that the average state in which the enzyme-1a and the enzyme-1a* complexes resided during the simulation time displayed a quite solvent-exposed active site, feasibly permitting esterification and hydrolysis processes to happen in situ. Thirdly, an examination of the distances between the domains responsible for active site closure indicated markedly reduced molecular motion of the enzyme-1a* complex compared to the enzyme-1a complex (Figs. and). While GtPyNP with bound 1a exhibited oscillatory opening and closing motions on a timescale of around 11 ns (similar dynamics are known for the closely related thymidine phosphorylases), binding of 1a* largely arrested this process. Specifically, binding of 1a* locks the enzyme in an open conformation by displacing a catalytically essential arginine residue, which reversibly adopts an inward position. This appears to halt domain movement and yield an inactive enzyme, while the esterification of the 2ꞌ-and 3ꞌ-OH groups of 1a further obstructs access to the anomeric carbon, collectively preventing productive phosphorolysis. As indicated by the clustered structures, 1a* also slightly obstructs access to the phosphate binding site of GtPyNP. Accordingly, we experimentally observed a decreased affinity for phosphate in the presence of 1a*, in addition to the lower kcat values stemming from pseudo-non-competitive inhibition by 1a* (Fig. ). Experimentally accessed and computed rate constants for the various processes involved in the catalytic cycle also proved consistent with the hypothesized mechanism. Enzyme opening/closing (ca. 0.1 ns -1 ) occurs on a much shorter time scale than the esterification of 1a (ca. 3 s -1 , Fig. ), catalysis (ca. 6 s -1 ) and substrate binding by GtPyNP (ca. 16 s -1 , see the SI). Thus, the comparably slow substrate release (ca. 0.2 s -1 ) necessitates in situ hydrolysis of 1a* to maintain the observed kinetics. In contrast to the phosphorolysis, the kinetics in the glycosylation direction remained essentially unchanged in the presence of borate (Figs. and), providing further support for the minor role of Rib1P* in this reaction system. Consistent with the entropically driven formation of 1a* from 1a, we observed decreased inhibition of the phosphorolysis reaction at higher temperatures, as evident from Eyring plots obtained with different borate concentrations (Fig. ). Taken together, these results support an inhibitory mechanism phenotypically resembling a non-competitive inhibition, where rapid equilibration of 1a to its borate ester 1a* (both in solution and while bound to the enzyme) reversibly decreases the fraction of catalytically active GtPyNP so that throughput in its catalytic cycle is primarily regulated by the position of the 1a-1a* equilibrium (Scheme 2). Preliminary data for other pyrimidine nucleoside phosphorylases as well as other nucleosides suggests that this inhibitory mechanism is likely not limited to GtPyNP and 1a (Figs. and). With a good understanding of the underlying processes governing the kinetics and thermodynamics of the phosphorolysis of 1a in the presence of borate, we aimed to apply the observed equilibrium shifts to other nucleosides, specifically targeting glycosylation reactions. Assuming that other nucleosides would behave in analogy to our model compound 1a, we expected that conversion shifts in glycosylation reactions would provide a general strategy to improve access to nucleosides from the precursor Rib1P (which could either be supplied as an isolated compound or generated in situ). Indeed, DFT calculations for a Scheme 2. Proposed mechanism for rate decreases in the presence of borate. representative set of nucleosides suggested that a variety of pyrimidine and purine nucleosides should undergo similar esterifications with borate as 1a (Table ), which could be confirmed by 1 H NMR (Δ ca. -32 to -34 kJ mol -1 , Fig. ) and, for two examples, with kinetic experiments (see the SI and Fig. for details). Although we were unable to translate the observed minor differences in Gibbs free energies to conversion shifts in transglycosylations, presumably due to a "kinetic lock" effect (see the SI and Fig. for details), glycosylation reactions with various pyrimidine nucleobases 2a-f nicely reflected the expected behavior and facilitated conversion shifts of 6-17% in favor of the respective nucleoside (Fig. ). A similar effect could be observed for the halogenated purine 2g when subjected to identical conditions with the promiscuous purine nucleoside phosphorylase from G. thermoglucosidasius. Lastly, an illustrative two-factor optimization for the glycosylation of 5-iodouracil (2d) and 5ethynyluracil (2f, both are known for their unfavorable glycosylation thermodynamics) showed how a balance of excess sugar donor and the "pull" effect of the corresponding nucleoside borate ester can be employed to improve the conversions in historically challenging nucleobase glycosylations (Fig. ). For instance, when using 2 eq. of Rib1P, the conversion of 2f to its nucleoside 1f could be improved from 74% to 89% through the application of 50 mM borate, which, conventionally, would have required the application of at least 6 eq. of Rib1P.