Mechanism of Action and Identification of Asp242 as the Catalytic Nucleophile of Vibrio furnisii N -Acetyl- β - d -glucosaminidase Using 2-Acetamido-2-deoxy-5-fluoro- α - l -idopyranosyl Fluoride
1999; American Chemical Society; Volume: 39; Issue: 1 Linguagem: Inglês
10.1021/bi991958d
ISSN1943-295X
AutoresDavid J. Vocadlo, Christoph Mayer, Shouming He, Stephen G. Withers,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-α-l-idopyranosyl fluoride has been synthesized, and the kinetic parameters Km = 0.23 mM and kcat = 0.55 min-1 for its hydrolysis by Vibrio furnisii β-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-β-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 neutral-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide, in conjunction with multiple sequence alignments of family 3 members, allowed the identification of Asp242 as the catalytic nucleophile within the sequence IVFSDDLSM.
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