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

Citation
Dj. Vocadlo et al., 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, BIOCHEM, 39(1), 2000, pp. 117-126
Citations number
48
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
1
Year of publication
2000
Pages
117 - 126
Database
ISI
SICI code
0006-2960(20000111)39:1<117:MOAAIO>2.0.ZU;2-A
Abstract
The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idop yranosyl 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 mixt ures of enzyme with this slow substrate by electrospray mass spectrometry r evealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro- beta-L-idopyranosyl-enzyme indicating that the hydrolytic mechanism of ExoI I involves the formation and rate-determining hydrolysis of a glycosyl-enzy me intermediate. Analysis of a peptic digest of the glycosyl-enzyme by HPLC /ESMS/MS in the neutral-loss mode permitted identification of a peptide bea ring the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide , in conjunction with multiple sequence alignments of family 3 members, all owed the identification of Asp242 as the catalytic nucleophile within the s equence IVFS (D) under bar DLSM.