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

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 Å resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid–base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032–16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2´,4´-dinitrophenyl 2-deoxy-2-fluoro-β-d-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters k_inact = 0.29±0.02 min^-1 and K_inact = 0.72±0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (K_i = 9±1 mM), a behaviour that was also observed with the substrate 2´,4´-dinitrophenyl β-d-cellobioside (k_cat = 40±1 s^-1, K_m = 0.35±0.03 mM, K_i = 24±4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (k_H2O = 0.0022 min^-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (k_react = 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 of this peptide by HPLC and subsequent collision-induced fragmentation by ESI–MS–MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid–base catalyst Glu-203 are conserved in all known family-12 sequences.