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 interac tion with the active site of an alpha -galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and s tructurally. The compound behaves as an apparently tight binding (K-i = 600 nM) competitive inhibitor, achieving this high affinity through reaction a s a slow substrate that accumulates a high steady-state concentration of th e glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the t rapped enzyme coupled with HPLC/MS analysis allowed the localization of a l abeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family reveal ed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenes is studies. (C) 2000 Elsevier Science Ltd. All rights reserved.