Description
Human GDP-L-fucose synthase (hGFS) converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose in three distinctive steps: two epimerisations at C3” and C5” positions of L-fucose sugar ring followed by NADPH-dependent reduction at C4”. Having only one isomeric product and complex reaction mechanism makes hGFS an extraordinary example of fine-tuned stereo selectivity in enzyme catalysed nucleotide sugar transformation. Currently the identity and precise roles of the acid/base residues that promote the catalysis is not fully understood. In this research we decided to probe hGFS through the prism of kinetic isotope effect, which is very powerful tool for determining all aspects of enzyme mechanisms, from kinetic to chemical mechanism and the structure of the transition state.For kinetic isotope effect measurements, we used combination of stopped flow rapid kinetic technic with spectrophotometric assay and synthesised deuterated substrates analogues: (4S)-[2H]-NADPH, [3”-2H]- and [5”-2H]-GDP-4-keto-6-deoxy-D-mannose. Measured KIEs for wild type hGFS points to a reaction in which the epimerisations pre-reduction are not rate-determining with KIEs ≃ 1. The reduction appears to be only partially rate-determining, judged by the KIE ≃ 1.4. “Burst phase” present from stopped flow experiment indicates that kcat is limited by release of the product (kobs ≃ 5.6 s-1, kcat ≃ 0.7 s-1). Measured KIEs for the active site mutants support the notion that Y143 residue involved in hydrid transfer during reduction step and C116/H186 base/acid pair involved in epimerisations.
| Period | 11 Jul 2023 |
|---|---|
| Event title | The 21st European Carbohydrate Symposium in Paris |
| Event type | Conference |
| Conference number | 21 |
| Location | Paris, FranceShow on map |
| Degree of Recognition | International |