@article { ISI:000085233200015,
	title = {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},
	journal = {BIOCHEMISTRY},
	volume = {39},
	number = {1},
	year = {2000},
	month = {JAN 11},
	pages = {117-126},
	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.},
	issn = {0006-2960},
	doi = {10.1021/bi991958d},
	author = {Vocadlo, DJ and Mayer, C and He, SM and Withers, SG}
}
