Identification of glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum beta-xylosidase using electrospray MS.
Thermoanaerobacterium saccharolyticum beta-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining beta-d-xylosidases and alpha-l-iduronidases. T. saccharolyticum beta-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl-enzyme intermediate. Incubation of T. saccharolyticum beta-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki=0.089 min-1, dissociation constant for the inactivator Ki=65 microM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-alpha-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.
Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
D J
DJ
Vocadlo
L F
LF
MacKenzie
S
S
He
G J
GJ
Zeikus
S G
SG
Withers
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D004789:Enzyme Activation; D018698:Glutamic Acid; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism*
D010446:Peptide Fragments; D018698:Glutamic Acid; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase