Prediction of kinetics and yields of nucleoside phosphorylase reactions and derived enzymatic reaction cascades

Reactions catalyzed by nucleoside phosphorylases are attractive routes towards the production of pharmaceutically interesting nucleoside compounds. Many different nucleoside phosphorylases were studied and applied for the production of nucleoside derivatives. Due to their broad substrate scope, the conversions of very different nucleoside and nucleobase compounds into each other were reported. However, the relationship between enzyme, compounds and reaction conditions in regard to the yield of reaction remained unclear so far.

The aim of this work is to understand this complex relationship. To achieve this, a high-throughput compatible UV/Vis assay for monitoring the nucleoside/nucleobase conversion was developed, and exhaustive experimental data was generated. From this data, a dynamic model of the kinetics of a pyrimidine nucleoside phosphorylase-catalyzed conversion of thymidine towards deoxyribose-1-phosphate was set up. The generated model indicated a fully reversible reaction, and thus the equilibrium state of this reaction was further investigated.

It was found that the reactions catalyzed by nucleoside phosphorylases behave strictly as reversible reactions. This means that the level of conversion in equilibrium state depends solely on intrinsic physical parameters, most importantly on the equilibrium constant. To be able to predict the yield of given reactions, the equilibrium constants for the phosphorolysis of 24 nucleoside compounds were determined. It was found that the Gibbs free energy of reaction from nucleosides towards pentose-1-phosphates is strictly positive in biologically relevant conditions. Furthermore, the Gibbs free energy of reaction which has to be overcome for production of pentose-1-phosphates is larger when starting from purine nucleosides, compared to pyrimidine nucleosides, though the transition between them is much smoother than expected. Varying the carbohydrate moiety between ribose and 2-deoxyribose did not influence the Gibbs free energy of reaction significantly.

The perception of nucleoside phosphorylase-catalyzed reactions as reversible reactions proceeding towards a pre-determined equilibrium conversion is elementary to the field. This understanding of thermodynamic reaction control will allow for the first time to conduct knowledge-driven reaction engineering on nucleoside phosphorylase-catalyzed reactions.