IDENTIFICATION OF GLU(340) AS THE ACTIVE-SITE NUCLEOPHILE IN HUMAN GLUCOCEREBROSIDASE BY USE OF ELECTROSPRAY TANDEM MASS-SPECTROMETRY

disease is an inherited lysosomal storage disorder caused by a deficie ncy of human acid beta-glucosidase (glucocerebrosidase). This enzyme i s inactivated by the specific, mechanism-based enzyme inactivator 2-de oxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by formin g a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate . The key nucleophilic amino acid residue involved in formation of thi s intermediate was conclusively identified by tandem mass spectrometry as Glu340, and not Asp443 as thought previously. This was confirmed b y site-directed mutagenesis. Identification, and mass determination, o f the labeled peptide in a proteolytic hydrolysate involved detection of a collision-induced fragmentation reaction specific to the sugar-pe ptide linkage. Confirmation of the identity of the labeled peptide was obtained both by tandem mass spectrometric sequencing and by chemical degradation of the purified peptide. This method allowed the rapid, s ensitive, and non-isotopic determination of an essential amino acid re sidue in a clinically important enzyme.