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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.

Mutations in GBA1, the gene for glucocerebrosidase (GCase), are genetic risk factors for Parkinson disease (PD). α-Synuclein (α-Syn), a protein implicated in PD, interacts with GCase and efficiently inhibits enzyme activity. GCase deficiency causes the lysosomal storage disorder Gaucher disease (GD). We show that saposin C (Sap C), a protein vital for GCase activity in vivo, protects GCase against α-syn inhibition. Using nuclear magnetic resonance spectroscopy, site-specific fluorescence, and Förster energy transfer probes, Sap C was observed to displace α-syn from GCase in solution and on lipid vesicles. Our results suggest that Sap C might play a crucial role in GD-related PD.

Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to β-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.

Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.