A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining β-glucosidases.

ABSTRACT: Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine

SUBMITTER: Kallemeijn WW 

PROVIDER: S-EPMC4271221 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature