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
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
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.