IDENTIFICATION OF GLUTAMIC-ACID-78 AS THE ACTIVE-SITE NUCLEOPHILE IN BACILLUS-SUBTILIS XYLANASE USING ELECTROSPRAY TANDEM MASS-SPECTROMETRY

A new mechanism-based inactivator of beta-1,4-xylanases, 2',4'-dinitro phenyl 2-deoxy-2-fluoro-beta-xylobioside, has been synthesized and use d to trap the covalent intermediate formed during catalysis by Bacillu s subtilis xylanase. Electrospray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of inactivator into the enzyme. Ina ctivation of xylanase followed the expected pseudo-first-order kinetic behavior, and kinetic parameters were determined. The intermediate tr apped was relatively stable toward hydrolytic turnover (t(1/2) = 350 m in). However, turnover could be facilitated by transglycosylation foll owing the addition of the acceptor benzyl thio-beta-xylobioside, thus demonstrating the catalytic competence of the trapped intermediate. Re activation kinetic parameters for this process of k(re) = 0.03 min(-1) and K-re = 46 mM were determined. The nucleophilic amino acid was ide ntified as Glu78 by a tandem mass spectrometric technique which does n ot require the use of radiolabels. The peptic digest of the labeled en zyme was separated by high-performance liquid chromatography and the e luent fed into a tandem mass spectrometer via an electrospray ionizati on device. The labeled peptide was identified as one of m/z = 826 (dou bly charged) which fragmented in the collision chamber between the mas s analyzers with loss of the mass of a 2-fluoroxylobiosyl unit. Confir mation of the peptide identity was obtained both by tandem mass spectr ometric sequencing and by Edman degradation of the purified peptide. G lu78 is completely conserved in all members of this xylanase family an d indeed is shown to be located in the active site in the recently det ermined X-ray crystal structure.