BROMOKETONE C-GLYCOSIDES, A NEW CLASS OF BETA-GLUCANASE INACTIVATORS

Although reliable methods have been developed for the labeling of the active site nucleophiles in glycosidases, no such reliable method has been developed for the identification of the acid/base catalyst. To ad dress this problem two novel bromoketone affinity labels based on a be ta-C-glucoside (6), and a beta-C-cellobioside (9), have been synthesiz ed via a chemoenzymatic process and tested as inactivators of the beta -glucosidase from Agrobacterium sp. and the beta-glucanases from Cellu lomonas fimi. The beta-glucosidase was inactivated by 6 in a time-depe ndent manner according to kinetic parameters of k(i) = 0.01 min(-1) an d K-I = 3.1 mM. Electrospray ionization mass spectrometric analysis re vealed that multiple labeling of the enzyme had occurred. The beta-end oglucanases, CenA and CenD, were inactivated stoicheometrically by 9 a ccording to kinetic parameters of k(i) = 0.0155 min(-1); K-I = 0.35 mM and k(i) = 0.01 min(-1); K-I = 6.0 mM, respectively. These should the refore prove to be valuable reagents for the labeling of glycosidases.