Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile.
Sweet almond beta-glucosidase is a well studied glycosidase, having been subjected to numerous kinetic analyses and inhibition studies. However, it is not known to which glycosidase family it belongs, nor is the identity of the active site nucleophile known with certainty. It can be inactivated using 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 glycosylated peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z = 1041 in the labeled sample. The sequence of this peptide was determined to be Ile-Thr-Glu-Gln-Gly-Val-Asp-Glu by further tandem mass spectrometric analysis in the daughter ion scan mode in conjunction with Edman degradation of the purified peptide. The identity of the labeled side chain was determined by further tandem mass spectrometric analysis in the daughter ion scan mode of a partially purified sample of the labeled peptide subjected to methyl esterification, the fragmentation pattern being consistent only with the first Glu in the sequence being labeled. The sequence around this residue is identical to that surrounding the catalytic nucleophile in many members of glycosidase Family 1, confirming the assignment of this enzyme to that family. The residue labeled is, however, different from that (Asp) identified previously in the enzyme from bitter almonds by use of conduritol epoxide affinity labels, although apparently close in the primary sequence.
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Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Withers
S G
SG
D000595:Amino Acid Sequence; D001665:Binding Sites; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D009754:Nuts / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*
D004791:Enzyme Inhibitors; C041022:2-deoxy-2-fluoroglucopyranosyl fluoride; D003847:Deoxyglucose; D001617:beta-Glucosidase