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

ABSTRACT: Retaining ?-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 ?-glucosidases. Whereas ?-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ?-glucosidases, ?-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ?-glucosidases by the combined use of cyclophellitol ?-epoxide- and ?-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent ?-aziridine but not ?-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ?-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ?-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ?-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

SUBMITTER: Kallemeijn WW 

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

REPOSITORIES: biostudies-literature