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Download fileHydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes
journal contribution
posted on 2016-07-25, 00:00 authored by Fredj Ben Bdira, Jianbing Jiang, Wouter Kallemeijn, Annett de Haan, Bogdan
I. Florea, Boris Bleijlevens, Rolf Boot, Herman S. Overkleeft, Johannes M. Aerts, Marcellus UbbinkSmall
compound active site interactors receive considerable attention
for their ability to positively influence the fold of glycosidases.
Endoglycoceramidase II (EGCII) from Rhodococcus sp.
is an endo-β-glucosidase releasing the complete glycan from
ceramide in glycosphingolipids. Cleavage of the β-glycosidic
linkage between glucose and ceramide is also catalyzed by glucocerebrosidase
(GBA), the exo-β-glucosidase deficient in Gaucher disease. We
demonstrate that established β-glucoside-configured cyclophellitol-type
activity-based probes (ABPs) for GBA also are effective, mechanism-based,
and irreversible inhibitors of EGCII. The stability of EGCII is markedly
enhanced by formation of covalent complexes with cyclophellitol ABPs
substituted with hydrophobic moieties, as evidenced by an increased
melting temperature, resistance against tryptic digestion, changes
in 15N–1H transverse relaxation optimized
spectroscopy spectra of the [15N]Leu-labeled enzyme, and
relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic
acid fluorescence. The stabilization of EGCII conformation correlates
with the shape and hydrophobicity of the substituents of the ABPs.
We conclude that the amphipathic active site binders with aliphatic
moieties act as a “hydrophobic zipper” on the flexible
EGCII protein structure.