|Title||IDENTIFICATION OF GLU(340) AS THE ACTIVE-SITE NUCLEOPHILE IN HUMAN GLUCOCEREBROSIDASE BY USE OF ELECTROSPRAY TANDEM MASS-SPECTROMETRY|
|Publication Type||Journal Article|
|Year of Publication||1994|
|Authors||MIAO, SC, MCCARTER, JD, GRACE, ME, GRABOWSKI, GA, AEBERSOLD, R, Withers, SG|
|Journal||JOURNAL OF BIOLOGICAL CHEMISTRY|
|Date Published||APR 15|
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.