The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean alpha-galactosidase and identify the catalytic nucleophile

5-Fluoro-alpha-D-galactopyranosyl fluoride was synthesized and its interaction with the active site of an alpha-galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding (Ki = 600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies.