title: A mass spectrometry-based approach for probing enzyme active sites: identification of Glu 127 in Cellulomonas fimi exoglycanase as the residue modified by N-bromoacetyl cellobiosylamine.
abstract: We have identified the residue in Cellulomonas fimi exoglycanase modified by N-bromoacetyl cellobiosylamine as Glu 127 using a new combination of experimental approaches. The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved with pepsin. The N-acetyl cellobiosylamine-modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chromatography connected online to an electrospray ionization mass spectrometer. The modified residue in the labeled peptide was determined by using a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry. Tandem mass spectrometry was used for further structural characterization of the cleaved residue. We show that the residue modified by N-bromoacetyl cellobiosylamine is Glu 127. This residue has been identified previously as the acid-base catalyst by using a combination of mutagenic and kinetic analyses. Our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residues in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current methods used to identify labeled residues within proteins.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver.
authors.forename: D
authors.initials: D
authors.lastname: Tull
authors.forename: D L
authors.initials: DL
authors.lastname: Burgoyne
authors.forename: D T
authors.initials: DT
authors.lastname: Chow
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002475:Cellobiose / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases; D018698:Glutamic Acid / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D014995:Xylosidases / Q000737:chemistry*; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D000345:Affinity Labels; C086529:N-bromoacetylcellobiosylamine; D002475:Cellobiose; D018698:Glutamic Acid; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase