Identification of Glu-540 as the catalytic nucleophile of human beta-glucuronidase using electrospray mass spectrometry

Human beta-glucuronidase is a member of the Family 2 glycosylhydrolases tha t cleaves beta-D-glucuronic acid residues from the nonreducing termini of g lycosaminoglycans. The enzyme is shown to catalyze glycoside bond hydrolysi s with net retention of anomeric configuration, presumably via a mechanism involving a covalent glucuronyl-enzyme intermediate. Incubation of human be ta-glucuronidase with 2-deoxy-2-fluoro-beta-D-glucuronyl fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme, as observed by electro spray mass spectrometry. Regeneration of the free enzyme by hydrolysis or t ransglycosylation and removal of excess inactivator demonstrated that the c ovalent intermediate was kinetically competent. Peptic digestion of the 2-d eoxy-2-fluoro-alpha-D-glucuronyl-enzyme intermediate and subsequent analysi s by liquid chromatography coupled with electrospray ionization triple quad rupole mass spectrometry indicated the presence of a 2-deoxy-2-fluoro-alpha -D-glucuronyl peptide. Sequence determination of the labeled peptide by tan dem mass spectrometry in the daughter ion scan mode permitted the identific ation of Glu-540 as the catalytic nucleophile within the sequence S (E) und er bar YGAET.