A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining ß-glucosidases.
J Biol Chem
; 289(51): 35351-62, 2014 Dec 19.
Article
em En
| MEDLINE
| ID: mdl-25344605
ABSTRACT
Retaining ß-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 ß-glucosidases. Whereas ß-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ß-glucosidases, ß-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ß-glucosidases by the combined use of cyclophellitol ß-epoxide- and ß-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 ß-aziridine but not ß-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ß-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ß-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ß-glucosidases categorized in GH1 and GH116 GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sondas Moleculares
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Beta-Glucosidase
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Cicloexanóis
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Aminoácidos
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2014
Tipo de documento:
Article