2. Academic Validation
  3. Hydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes

Hydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes

  • Biochemistry. 2016 Aug 30;55(34):4823-35. doi: 10.1021/acs.biochem.6b00363.
Fredj Ben Bdira 1,  Jianbing Jiang 2,  Wouter Kallemeijn 3,  Annett de Haan 4,  Bogdan I Florea 2,  Boris Bleijlevens 4,  Rolf Boot 3,  Herman S Overkleeft 2,  Johannes M Aerts 3,  Marcellus Ubbink 1
Affiliations

Affiliations

  • 1 Department of Macromolecular Biochemistry, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
  • 2 Department of Bio-organic Synthesis, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
  • 3 Department of Medical Biochemistry, Leiden Institute of Chemistry , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
  • 4 Department of Medical Biochemistry, Academic Medical Center , Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
PMID: 27455091 DOI: 10.1021/acs.biochem.6b00363
Abstract

Small 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 (15)N-(1)H transverse relaxation optimized spectroscopy spectra of the [(15)N]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.

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