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

TitleIdentification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB
Publication TypeJournal Article
Year of Publication1998
AuthorsZechel, DL, He, SM, Dupont, C, Withers, SG
JournalBIOCHEMICAL JOURNAL
Volume336
Pagination139-145
Date PublishedNOV 15
ISSN0264-6021
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',4'-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',4'-dinitrophenyl beta-D-cellobioside (k(cat) = 40+/-1 s(-1)