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

TitleIDENTIFICATION OF GLU(340) AS THE ACTIVE-SITE NUCLEOPHILE IN HUMAN GLUCOCEREBROSIDASE BY USE OF ELECTROSPRAY TANDEM MASS-SPECTROMETRY
Publication TypeJournal Article
Year of Publication1994
AuthorsMIAO, SC, MCCARTER, JD, GRACE, ME, GRABOWSKI, GA, AEBERSOLD, R, Withers, SG
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume269
Pagination10975-10978
Date PublishedAPR 15
ISSN0021-9258
Abstract

disease is an inherited lysosomal storage disorder caused by a deficiency of human acid beta-glucosidase (glucocerebrosidase). This enzyme is inactivated by the specific, mechanism-based enzyme inactivator 2-deoxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by forming a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate. The key nucleophilic amino acid residue involved in formation of this intermediate was conclusively identified by tandem mass spectrometry as Glu340, and not Asp443 as thought previously. This was confirmed by site-directed mutagenesis. Identification, and mass determination, of the labeled peptide in a proteolytic hydrolysate involved detection of a collision-induced fragmentation reaction specific to the sugar-peptide 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, sensitive, and non-isotopic determination of an essential amino acid residue in a clinically important enzyme.