@article { ISI:000077255100017,
	title = {Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB},
	journal = {BIOCHEMICAL JOURNAL},
	volume = {336},
	number = {1},
	year = {1998},
	month = {NOV 15},
	pages = {139-145},
	abstract = {{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 Angstrom 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{\textquoteright},4{\textquoteright}-dinitrophenyl 2-deoxy-2-fluoro-beta-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{\textquoteright},4{\textquoteright}-dinitrophenyl beta-D-cellobioside (k(cat) = 40+/-1 s(-1)},
	issn = {0264-6021},
	author = {Zechel, DL and He, SM and Dupont, C and Withers, SG}
}
