IDENTIFICATION OF GLU-277 AS THE CATALYTIC NUCLEOPHILE OF THERMOANAEROBACTERIUM-SACCHAROLYTICUM BETA-XYLOSIDASE USING ELECTROSPRAY MS

Thermoanaerobacterium saccharolyticum beta-xylosidase is a member of f amily 39 of the glycosyl hydrolases. This grouping comprises both reta ining beta-D-xylosidases and alpha-L-iduronidases. T. saccharolyticum beta-xylosidase catalyses the hydrolysis of short xylo-oligosaccharide s into free xylose via a covalent xylosyl-enzyme intermediate. Incubat ion of T. saccharolyticum beta-xylosidase with 2,4-dinitrophenyl 2-deo xy-2-fluoro-beta-D-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(perpendicular to)= 0.089 min (-1), dissociation constant for the inactivator K-perpendicular to = 6 5 mu M) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha- D-xylosyl-enzyme, as observed by electrospray MS. Removal of excess in activator and regeneration of the free enzyme through transglycosylati on with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of t he 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutr al-loss mode indicated the presence of a 2-deoxy-2-fluoro-alpha-D-xylo syl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-2 77 (bold and underlined) as the catalytic nucleophile within the seque nce IILNSHFPNLPFHIT (E) under bar Y.