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 that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. The enzyme is shown to catalyze glycoside bond hydrolysis with net retention of anomeric configuration, presumably via a mechanism involving a covalent glucuronyl-enzyme intermediate. Incubation of human beta-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 electrospray mass spectrometry. Regeneration of the free enzyme by hydrolysis or transglycosylation and removal of excess inactivator demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme intermediate and subsequent analysis by liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry indicated the presence of a 2-deoxy-2-fluoro-alpha-D-glucuronyl peptide. Sequence determination of the labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Glu-540 as the catalytic nucleophile within the sequence SEYGAET.
Wong
A W
AW
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
He
S
S
Grubb
J H
JH
Sly
W S
WS
Withers
S G
SG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D017124:Conserved Sequence; D005960:Glucosides / Q000138:chemical synthesis / Q000737:chemistry; D005966:Glucuronidase / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D005960:Glucosides; D010446:Peptide Fragments; D018698:Glutamic Acid; D005966:Glucuronidase