Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-beta-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl fluoride.
The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosykl fluoride has been synthesized, and the kinetic parameters K(M) = 0.23 mM and K(CAT)= 0.55 min(-1) for its hydrolysis by vibrio furnisi beta-N-acetylglucosaminidase (ExoII) HAVE been determined. Investigation of mixtures of enzyme with this slow substrate by electrospray mass spectrometry revealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro-beta-L-idopyranosyl-enzyme, indicating that the hydrolytic mechanism of ExoII involves the formation and rate-determining hydrolysis of a glycosyl-enzyme intermediate. Analysis of a peptic digest of the glycosyl-enzyme by HPLC/ESMS/MS in the netural-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide, in conjuction with multiple sequence alignmentsS of family 3 members, allowed the identification of ASP242 as the catalytic nucleophile within the sequence IVFSDDLSM.
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Protein Engineering Network of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver.
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D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D009005:Monosaccharides / Q000737:chemistry*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014733:Vibrio / Q000201:enzymology*
C403207:2-acetamido-2-deoxy-5-fluoro-idopyranosyl fluoride; D007202:Indicators and Reagents; D009005:Monosaccharides; D010455:Peptides; D001224:Aspartic Acid; D000118:Acetylglucosaminidase