Crystal Structures of Complexes of N-Butyl- and N-Nonyl-Deoxynojirimycin Bound to Acid β-Glucosidase

Abstract

Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars. Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars. Mutations in the gene encoding acid β-glucosidase (GlcCerase 3The abbreviations used are: GlcCeraseacid β-glucosidaseCBEconduritol-B-epoxideendo-GCase IIendo-glycoceramidase IIGlcCerglucosylceramideIFGisofagomineNB-DNJN-butyl-deoxynojirimycinNN-DNJN-nonyl-deoxynojirimycinGM3NeuAcα 2,3Galβ1,4Glc-ceramideDGdeglycosylated. 3The abbreviations used are: GlcCeraseacid β-glucosidaseCBEconduritol-B-epoxideendo-GCase IIendo-glycoceramidase IIGlcCerglucosylceramideIFGisofagomineNB-DNJN-butyl-deoxynojirimycinNN-DNJN-nonyl-deoxynojirimycinGM3NeuAcα 2,3Galβ1,4Glc-ceramideDGdeglycosylated., E.C. 3.2.1.45) cause Gaucher disease, the most common lysosomal storage disorder (1Futerman A.H. Zimran A. Gaucher Disease. Taylor and Francis Group, Boca Raton, FL2006Crossref Google Scholar, 2Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (618) Google Scholar, 3Beutler E. Grabowski G.A. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar, 4Jmoudiak M. Futerman A.H. Br. J. Haematol. 2005; 129: 178-188Crossref PubMed Scopus (224) Google Scholar, 5Premkumar Futerman A.H. J. Biol. 2005; PubMed Scopus Google GlcCerase the of glucosylceramide (GlcCer) to and G.A. M. Rev. Mol. Biol. PubMed Scopus Google of the of GlcCerase that M. Futerman A.H. PubMed Scopus Google of a by a and of is a the catalytic site M. Futerman A.H. PubMed Scopus Google to understanding of the GlcCerase B. Futerman A.H. Biol. PubMed Scopus Google and to of GlcCerase to which Futerman A.H. J. Biol. 2005; PubMed Scopus Google D. G.A. Nat. Biol. PubMed Scopus Google of are that cause a of in of catalytic M. PubMed Scopus Google Scholar, M. D. Biol. PubMed Scopus Google to and the abbreviations used in the to human expressed in GlcCerase expressed in plant GlcCerase expressed in plant GlcCerase expressed in plant human expressed in M. Futerman A.H. PubMed Scopus Google D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google in a are available for Gaucher disease The is recombinant human GlcCerase expressed in A.H. M. Zimran A. 2004; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. 2004; PubMed Scopus Google The is in which of by N-butyl-deoxynojirimycin J. Biol. PubMed Google of is on the chaperone in which active inhibitors are used to of GlcCerase as they the PubMed Scopus Google Scholar, PubMed Scopus Google NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) also as chemical of GlcCerase 2005; PubMed Scopus Google GlcCerase of cultured GlcCerase with NN-DNJ NB-DNJ E. A. PubMed Scopus Google Scholar, J. J. M. Mol. 2005; PubMed Scopus Google NB-DNJ and NN-DNJ inhibit GlcCer J. Biol. PubMed Google and GlcCerase, although at different 2005; PubMed Scopus Google Scholar, E. A. PubMed Scopus Google The mechanism of of to GlcCerase, is now report the crystal of complexes of NB-DNJ and NN-DNJ with recombinant human GlcCerase, expressed in cultured plant D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google the of is to that of D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google We the of to NB-DNJ and NN-DNJ with that of a of with and in mode of that have for understanding the catalytic of GlcCerase and the mode of chaperoning by imino as D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The in and to in a a with a NB-DNJ and NN-DNJ in water to and to the to a concentration of the and and that the and The of in and the of with for and on the of the with the and with Scopus Google The of and and with Biol. PubMed Scopus Google The crystal of D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google used as a and the and water Biol. 2004; PubMed Scopus Google with and PubMed Scopus Google and in the for and for and of of in a of imino sugar of NN-DNJ used to a of GlcCer in the active site. the of NN-DNJ the the acid of the moiety on the of the crystal of the of with J. Biol. PubMed Scopus Google The catalytic of with of GlcCerase of to of GlcCerase, and of to of in of with of The moiety of GlcCer the imino sugar of the of GlcCer the of the GlcCer moiety of to the of the and of bonds of the to of NB-DNJ and NN-DNJ to the of of and in with in the of the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google on of in of only and that of these a to the inhibitors bind to the active site of with the imino sugar making hydrogen bonds with side chains of active site and the imino sugar and the active site are for NB-DNJ and The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site. The of with these alkyl chains are hydrophobic and than the hydrogen with the with the of imino with alkyl chains of to bind to GlcCerase 2005; PubMed Scopus Google The alkyl chains of inhibitors are by with the active and the alkyl of NN-DNJ with the entrance to the active site is a in the of the inhibitors for NB-DNJ and for A. G. PubMed Scopus Google which for by the of NN-DNJ is most a of the of NN-DNJ with NB-DNJ with the hydrophobic at the entrance to the active site the hydrophobic of of inhibitors to hydrogen bonds and hydrophobic interactions. in to the side of a of inhibitors to the active site of NB-DNJ and NN-DNJ to to D. G.A. Nat. Biol. PubMed Scopus Google chains of the in are as in in and in and of bonds in of GlcCerase of of with of for molecule in of a to a of for molecule in a to a in a of the of and with that of D. G.A. Nat. Biol. PubMed Scopus Google that the a number of hydrogen bonds with the in and these is in the of with the active site of The in is to the of GlcCer and is located the catalytic acid and Glu-340, hydrogen bonds with of the of NN-DNJ and NB-DNJ are located in a to that of the of GlcCer and hydrogen bonds with at the to the active site of GlcCerase is on side by which have in a number of and Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, M. Futerman A.H. PubMed Scopus Google Scholar, B. Futerman A.H. Biol. PubMed Scopus Google Scholar, B. A. M. C.R. Grabowski G.A. J. Biol. PubMed Scopus Google in GlcCerase to which only of Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, D. G.A. Nat. Biol. PubMed Scopus Google which the as that in in D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The of the active loops the side of on a hydrogen with the that to the on the of GlcCer in a in a of the loops at the entrance to the active site. The loops in GlcCerase in a number of only are for in and these the most in the entrance to the active site. which a role in the catalytic is The catalytic are as on that to is the to which a is and the of hydrogen bonds to to in a the with a in which is to Glu-340, a hydrogen with and a the of are by the a in of the GlcCerase in in to by crystal B. Futerman A.H. Biol. PubMed Scopus Google and in are the on and are in crystal and is that is in The of these hydrophobic to the of GlcCerase with the B. Futerman A.H. Biol. PubMed Scopus Google of on the and on that of to the active site of J. Biol. PubMed Scopus Google we GlcCer in the active site of GlcCerase to the mechanism of of of of inhibitors to GlcCerase with of GlcCer to a of GlcCer is on the of NN-DNJ the active site are in and are as and NN-DNJ in the active site of of the GlcCer moiety of the active site of of GlcCer in the active site of is to a nucleophilic role for in the catalytic G. B. PubMed Scopus Google have attack of the of on the of The and a the hydrogen on the is the and the in a that nucleophilic attack by is a attack by G. PubMed Scopus Google Scholar, Rev. Scopus Google the of nucleophilic attack on the by is the hydrogen is the to catalytic mechanism of the of to nucleophilic attack by to with the hydrogen of the of the a the mechanism a nucleophilic attack on the of a the of to in which a to the in the of the and thus in the of the G. PubMed Scopus Google Scholar, Rev. Scopus Google The of the of with a of is to of The of in by partially GlcCerase a with of and J. PubMed Google Scholar, G. M. Grabowski G.A. PubMed Scopus Google in B. Futerman A.H. Biol. PubMed Scopus Google is and is of a hydrogen and the catalytic to is in the active site. The of is only to the of than to is thus required to the of these hydrogen and and are The of the moiety of is to the of the role of water in the catalytic a water molecule is required for of the GlcCerase of to water and a cavity the catalytic water water molecule at is in structures, as J. Biol. PubMed Scopus Google and J. A. PubMed Scopus Google conserved water in the catalytic the solvent cavity is located to the catalytic and is the active site also site. of the of we a by the side chains of and to the is that the with the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google is by a a in the GlcCerase and site as a site of GlcCerase with the B. Futerman A.H. Biol. PubMed Scopus Google solvent adjacent to catalytic The water molecule common to is by is the only currently available orally administered for treatment of Gaucher is of GlcCer the also a chaperone on The chaperoning of NN-DNJ to for GlcCerase with mutations in the catalytic M. D. Biol. PubMed Scopus Google Scholar, E. A. PubMed Scopus Google Scholar, E. Biol. 2005; PubMed Scopus Google The crystal of and are with these the at the active site. The of NB-DNJ also to for role as a the where the is the is to the of these NB-DNJ and to the active site. at the of the NB-DNJ thus and the of thus with Mutations in the gene encoding acid β-glucosidase (GlcCerase 3The abbreviations used are: GlcCeraseacid β-glucosidaseCBEconduritol-B-epoxideendo-GCase IIendo-glycoceramidase IIGlcCerglucosylceramideIFGisofagomineNB-DNJN-butyl-deoxynojirimycinNN-DNJN-nonyl-deoxynojirimycinGM3NeuAcα 2,3Galβ1,4Glc-ceramideDGdeglycosylated. 3The abbreviations used are: GlcCeraseacid β-glucosidaseCBEconduritol-B-epoxideendo-GCase IIendo-glycoceramidase IIGlcCerglucosylceramideIFGisofagomineNB-DNJN-butyl-deoxynojirimycinNN-DNJN-nonyl-deoxynojirimycinGM3NeuAcα 2,3Galβ1,4Glc-ceramideDGdeglycosylated., E.C. 3.2.1.45) cause Gaucher disease, the most common lysosomal storage disorder (1Futerman A.H. Zimran A. Gaucher Disease. Taylor and Francis Group, Boca Raton, FL2006Crossref Google Scholar, 2Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (618) Google Scholar, 3Beutler E. Grabowski G.A. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar, 4Jmoudiak M. Futerman A.H. Br. J. Haematol. 2005; 129: 178-188Crossref PubMed Scopus (224) Google Scholar, 5Premkumar Futerman A.H. J. Biol. 2005; PubMed Scopus Google GlcCerase the of glucosylceramide (GlcCer) to and G.A. M. Rev. Mol. Biol. PubMed Scopus Google of the of GlcCerase that M. Futerman A.H. PubMed Scopus Google of a by a and of is a the catalytic site M. Futerman A.H. PubMed Scopus Google acid β-glucosidase glucosylceramide N-butyl-deoxynojirimycin N-nonyl-deoxynojirimycin acid β-glucosidase glucosylceramide N-butyl-deoxynojirimycin N-nonyl-deoxynojirimycin to understanding of the GlcCerase B. Futerman A.H. Biol. PubMed Scopus Google and to of GlcCerase to which Futerman A.H. J. Biol. 2005; PubMed Scopus Google D. G.A. Nat. Biol. PubMed Scopus Google of are that cause a of in of catalytic M. PubMed Scopus Google Scholar, M. D. Biol. PubMed Scopus Google are available for Gaucher disease The is recombinant human GlcCerase expressed in A.H. M. Zimran A. 2004; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. 2004; PubMed Scopus Google The is in which of by N-butyl-deoxynojirimycin J. Biol. PubMed Google of is on the chaperone in which active inhibitors are used to of GlcCerase as they the PubMed Scopus Google Scholar, PubMed Scopus Google NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) also as chemical of GlcCerase 2005; PubMed Scopus Google GlcCerase of cultured GlcCerase with NN-DNJ NB-DNJ E. A. PubMed Scopus Google Scholar, J. J. M. Mol. 2005; PubMed Scopus Google NB-DNJ and NN-DNJ inhibit GlcCer J. Biol. PubMed Google and GlcCerase, although at different 2005; PubMed Scopus Google Scholar, E. A. PubMed Scopus Google The mechanism of of to GlcCerase, is We now report the crystal of complexes of NB-DNJ and NN-DNJ with recombinant human GlcCerase, expressed in cultured plant D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google the of is to that of D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google We the of to NB-DNJ and NN-DNJ with that of a of with and in mode of that have for understanding the catalytic of GlcCerase and the mode of chaperoning by imino sugars. as D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The in and to in a a with a NB-DNJ and NN-DNJ in water to and to the to a concentration of the and and that the and The of in and the of with for and on the of the with the and with Scopus Google The of and and with Biol. PubMed Scopus Google The crystal of D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google used as a and the and water Biol. 2004; PubMed Scopus Google with and PubMed Scopus Google and in the for and for and of of in a of imino sugar of NN-DNJ used to a of GlcCer in the active site. the of NN-DNJ the the acid of the moiety on the of the crystal of the of with J. Biol. PubMed Scopus Google The catalytic of with of GlcCerase of to of GlcCerase, and of to of in of with of The moiety of GlcCer the imino sugar of the of GlcCer the of the GlcCer moiety of to the of the and of bonds of the to as D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The in and to in a a with a NB-DNJ and NN-DNJ in water to and to the to a concentration of the and and that the and The of in and the of with for and on the of the with the and with Scopus Google The of and and with Biol. PubMed Scopus Google The crystal of D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google used as a and the and water Biol. 2004; PubMed Scopus Google with and PubMed Scopus Google and in the for and for of imino sugar of NN-DNJ used to a of GlcCer in the active site. the of NN-DNJ the the acid of the moiety on the of the crystal of the of with J. Biol. PubMed Scopus Google The catalytic of with of GlcCerase of to of GlcCerase, and of to of in of with of The moiety of GlcCer the imino sugar of the of GlcCer the of the GlcCer moiety of to the of the and of bonds of the to of NB-DNJ and NN-DNJ to the of of and in with in the of the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google on of in of only and that of these a to the inhibitors bind to the active site of with the imino sugar making hydrogen bonds with side chains of active site and the imino sugar and the active site are for NB-DNJ and The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site. The of with these alkyl chains are hydrophobic and than the hydrogen with the with the of imino with alkyl chains of to bind to GlcCerase 2005; PubMed Scopus Google The alkyl chains of inhibitors are by with the active and the alkyl of NN-DNJ with the entrance to the active site is a in the of the inhibitors for NB-DNJ and for A. G. PubMed Scopus Google which for by the of NN-DNJ is most a of the of NN-DNJ with NB-DNJ with the hydrophobic at the entrance to the active site the hydrophobic of inhibitors to the active site of NB-DNJ and NN-DNJ to to D. G.A. Nat. Biol. PubMed Scopus Google chains of the in are as in in and in and of bonds in of GlcCerase of of with of for molecule in of a to a of for molecule in a to a in a of the of and with that of D. G.A. Nat. Biol. PubMed Scopus Google that the a number of hydrogen bonds with the in and these is in the of with the active site of The in is to the of GlcCer and is located the catalytic acid and Glu-340, hydrogen bonds with of the of NN-DNJ and NB-DNJ are located in a to that of the of GlcCer and hydrogen bonds with at the to the active site of GlcCerase is on side by which have in a number of and Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, M. Futerman A.H. PubMed Scopus Google Scholar, B. Futerman A.H. Biol. PubMed Scopus Google Scholar, B. A. M. C.R. Grabowski G.A. J. Biol. PubMed Scopus Google in GlcCerase to which only of Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, D. G.A. Nat. Biol. PubMed Scopus Google which the as that in in D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The of the active loops the side of on a hydrogen with the that to the on the of GlcCer in a in a of the loops at the entrance to the active site. The loops in GlcCerase in a number of only are for in and these the most in the entrance to the active site. which a role in the catalytic is The catalytic are as on that to is the to which a is and the of hydrogen bonds to to in a the with a in which is to Glu-340, a hydrogen with and a the of are by the a in of the GlcCerase in in to by crystal B. Futerman A.H. Biol. PubMed Scopus Google and in are the on and are in crystal and is that is in The of these hydrophobic to the of GlcCerase with the B. Futerman A.H. Biol. 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Scopus Google The of the of with a of is to of The of in by partially GlcCerase a with of and J. PubMed Google Scholar, G. M. Grabowski G.A. PubMed Scopus Google in B. Futerman A.H. Biol. PubMed Scopus Google is and is of a hydrogen and the catalytic to is in the active site. The of is only to the of than to is thus required to the of these hydrogen and and are The of the moiety of is to the of the role of water in the catalytic a water molecule is required for of the GlcCerase of to water and a cavity the catalytic water water molecule at is in structures, as J. Biol. PubMed Scopus Google and J. A. PubMed Scopus Google conserved water in the catalytic the solvent cavity is located to the catalytic and is the active site also site. of the of we a by the side chains of and to the is that the with the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google is by a a in the GlcCerase and site as a site of GlcCerase with the B. Futerman A.H. Biol. PubMed Scopus Google solvent adjacent to catalytic The water molecule common to is by of NB-DNJ and NN-DNJ to the of of and in with in the of the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google on of in of only and that of these a to the Both inhibitors bind to the active site of with the imino sugar making hydrogen bonds with side chains of active site and the imino sugar and the active site are for NB-DNJ and The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site. The of with these alkyl chains are hydrophobic and than the hydrogen with the with the of imino with alkyl chains of to bind to GlcCerase 2005; PubMed Scopus Google The alkyl chains of inhibitors are by with the active and the alkyl of NN-DNJ with the entrance to the active site is a in the of the inhibitors for NB-DNJ and for A. G. PubMed Scopus Google which for by the of NN-DNJ is most a of the of NN-DNJ with NB-DNJ with the hydrophobic at the entrance to the active site the hydrophobic of the of and with that of D. G.A. Nat. Biol. PubMed Scopus Google that the a number of hydrogen bonds with the in and these is in the of with the active site of The in is to the of GlcCer and is located the catalytic acid and Glu-340, hydrogen bonds with of the of NN-DNJ and NB-DNJ are located in a to that of the of GlcCer and hydrogen bonds with at the to the active site of GlcCerase is on side by which have in a number of and Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, M. Futerman A.H. PubMed Scopus Google Scholar, B. Futerman A.H. Biol. PubMed Scopus Google Scholar, B. A. M. C.R. Grabowski G.A. J. Biol. PubMed Scopus Google in GlcCerase to which only of Futerman A.H. J. Biol. 2005; PubMed Scopus Google Scholar, D. G.A. Nat. Biol. PubMed Scopus Google which the as that in in D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google The of the active loops the side of on a hydrogen with the that to the on the of GlcCer in a in a on that to is the to which a is and the of hydrogen bonds to to in a the with a in which is to Glu-340, a hydrogen with and a the of are by the a in of the GlcCerase in in to by crystal B. Futerman A.H. Biol. PubMed Scopus Google and in are the on and are in crystal and is that is in The of these hydrophobic to the of GlcCerase with the B. Futerman A.H. Biol. PubMed Scopus Google of on the and on that of to the active site of J. Biol. PubMed Scopus Google we GlcCer in the active site of GlcCerase to the mechanism of of is to a nucleophilic role for in the catalytic G. B. PubMed Scopus Google have attack of the of on the of The and a the hydrogen on the is the and the in a that nucleophilic attack by is a attack by G. PubMed Scopus Google Scholar, Rev. Scopus Google the of nucleophilic attack on the by is the hydrogen is the to The the of to in which a to the in the of the and thus in the of the G. PubMed Scopus Google Scholar, Rev. Scopus Google The of the of with a of is to of The of in by partially GlcCerase a with of and J. PubMed Google Scholar, G. M. Grabowski G.A. PubMed Scopus Google in B. Futerman A.H. Biol. PubMed Scopus Google is and is of a hydrogen and the catalytic to is in the active site. The of is only to the of than to is thus required to the of these residues. The the role of water in the catalytic a water molecule is required for of the GlcCerase of to water and a cavity the catalytic water water molecule at is in structures, as J. Biol. PubMed Scopus Google and J. A. PubMed Scopus Google conserved water in the catalytic the solvent cavity is located to the catalytic and is the active site also site. of the of we a by the side chains of and to the is that the with the D. G. E. G. Futerman A.H. D. J. 5: PubMed Scopus Google is by a a in the GlcCerase and site as a site of GlcCerase with the B. Futerman A.H. Biol. PubMed Scopus Google is the only currently available orally administered for treatment of Gaucher is of GlcCer the also a chaperone on The chaperoning of NN-DNJ to for GlcCerase with mutations in the catalytic M. D. Biol. PubMed Scopus Google Scholar, E. A. PubMed Scopus Google Scholar, E. Biol. 2005; PubMed Scopus Google The crystal of and are with these the at the active site. The of NB-DNJ also to for role as a the where the is the is to the of these NB-DNJ and to the active site. at the of the NB-DNJ thus and the of thus with NB-DNJ is the only currently available orally administered for treatment of Gaucher is of GlcCer the also a chaperone on The chaperoning of NN-DNJ to for GlcCerase with mutations in the catalytic M. D. Biol. PubMed Scopus Google Scholar, E. A. PubMed Scopus Google Scholar, E. Biol. 2005; PubMed Scopus Google The crystal of and are with these the at the active site. The of NB-DNJ also to for role as a the where the is the is to the of these NB-DNJ and to the active site. at the of the NB-DNJ thus and the of thus with