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摘要
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.
- 出版日期2014-12-19
