|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|
|Publication Type||Journal Article|
|Year of Publication||2000|
|Authors||Vocadlo, DJ, Mayer, C, He, SM, Withers, SG|
|Date Published||JAN 11|
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.