Kinetic Examination and Simulation of GDP-ß-L-fucose Synthetase Reaction Using NADPH or NADH

GDP-beta-L -fucose synthetase (GFS) is a unique, bifunctional enzyme belonging to the family of short chain dehydrogenases (SDRs), which catalyses the conversion of GDP-4-keto-6-deoxy-alpha-D-mannose (GKDM) to GDP-beta-L-fucose (GDP-Fuc). A facile method for preparing GKDM in a stable and storable form on a 200 mumol scale was established. A simple macroscopic kinetic model has been used to produce a tool for simulation of several GFS reactions in batch mode. The respective kinetic parameters were determined considering GFS using NADPH and NADH as cofactor, demonstrating that GFS has a different affinity for GKDM depending on the cofactor. A comparison of simulated and experimentally performed batch reactions revealed that the GFS reaction can be described by this simple kinetic model that takes into account product inhibition by GDP-Fuc and NADP or NAD. Full H-1- and C-13-NMR characterisation of GKDM unambiguously showed that this compound exists in two different forms in aqueous solution, which we identified as the ketone and the respective hydrate form of GKDM. In equilibrium these species coexist in a ratio of about 70:30 (keto/hydrate form)