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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

TitleMechanism 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
Publication TypeJournal Article
Year of Publication2000
AuthorsVocadlo, DJ, Mayer, C, He, SM, Withers, SG
JournalBIOCHEMISTRY
Volume39
Pagination117-126
Date PublishedJAN 11
ISSN0006-2960
Abstract

The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl 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 furnisii 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 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 IVFS (D) under bar DLSM.

DOI10.1021/bi991958d