Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cel lulose with retention of anomeric configuration. A recent X-ray structure o f the catalytic domain at 1.75 Angstrom resolution has led to the prelimina ry assignment of Glu-120 and Glu-203 as the catalytic nucleophile and gener al acid-base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davi es (1997) Biochemistry 36, 16032-16039]. The present study confirms the ide ntity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D -cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturab le fashion, yielding the parameters k(inact) = 0.29+/-0.02 min(-1) and K-in act = 0.72 +/- 0.08 mM. Uncompetitive inhibition was observed at high conce ntrations of 2FDNPC (K-i = 9+/-1 mM), a behaviour that was also observed wi th the substrate 2',4'-dinitrophenyl beta-D-cellobioside (k(cat) = 40+/-1 s (-1), K-m = 0.35+/-0.03 mM, K-i = 24+/-4 mM). Protection against inactivati on was afforded by the competitive inhibitor cellobiose. The electrospray i onization (ESI) mass spectrum of the intact labelled CelB indicated that th e inactivator had labelled the enzyme stoichiometrically. Reactivation of t he trapped intermediate occurred spontaneously (k(H2O) = 0.0022 min(-1)) or via transglycosylation, with cellobiose acting as an acceptor ligand (k(re act) = 0.024 min(-1), K-react = 54 mM). Digestion of the labelled enzyme by pepsin followed by LC-ESI-tandem MS (MS-MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation o f this peptide by HPLC and subsequent collision-induced fragmentation by ES I-MS-MS produced a daughter-ion spectrum that corresponded to a sequence (Q TEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid-bas e catalyst Glu-203 are conserved in all known family-12 sequences.