Biochemical and Structural Assessment of the 1-N-Azasugar GalNAc-isofagomine as a Potent Family 20 β-N-Acetylhexosaminidase Inhibitor

Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining β-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the “anomeric” center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining β-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatusβ-N-acetylhexos-aminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to “attack” the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2 R,3 R,4 S,5 R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine·HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (Ki= 2.7 μm). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 Å and revealed that the lack of a strong electrostatic interaction between the “anomeric” center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-Å hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX. Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining β-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the “anomeric” center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining β-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatusβ-N-acetylhexos-aminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to “attack” the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2 R,3 R,4 S,5 R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine·HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (Ki= 2.7 μm). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 Å and revealed that the lack of a strong electrostatic interaction between the “anomeric” center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-Å hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX. Streptomyces plicatusβ-hexosaminidase β-N-acetylhexosaminidase Serratia marcescens chitobiase N-acetylglucosamine-thiazoline N-acetyl-galactosamine (2 R,3 R,4 S,5 R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside Natural and synthetic glycosidase inhibitors are useful biological tools for helping to understand the catalytic mechanism by which these ubiquitous enzymes process their natural substrates. Exploring the relationships between inhibitor structure, enzyme kinetics, and the molecular interactions that occur between an inhibitor and its enzyme target, not only provides insight into the catalytic mechanism of these enzymes but also gives an opportunity for knowledge-based design of potent and highly specific therapeutic agents (1Heightman T.D. Vasella A.T. Angew. Chem. Int. Ed. Engl. 1999; 38: 750-770Crossref PubMed Google Scholar). Indeed, glycosidases have been implicated in numerous carbohydrate-mediated processes related to disease, and much effort has been devoted to selectively controlling such glycosidase activity. Prominent examples include the use of sialidase inhibitors such as zanamivir (Relenza), oseltamivir (Tamiflu), and the experimental compound BCX-1812 for the treatment of influenza (2Gubareva L.V. Kaiser L. Hayden F.G. Lancet. 2000; 355: 827-835Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 3Taylor G. Curr. Opin. Struct. Biol. 1996; 6: 830-837Crossref PubMed Scopus (209) Google Scholar) and intestinal α-glucosidase inhibitors (e.g. acarbose and miglitol) for the treatment of non-insulin-dependent diabetes mellitus (4Martin A.E. Montgomery P.A. Am. J. Health Syst. Pharm. 1996; 53: 2277-2290Crossref PubMed Scopus (103) Google Scholar, 5Welborn T.A. Med. J. Aust. 1998; 168: 76-78Crossref PubMed Scopus (5) Google Scholar). The recent successes of sugar-based therapeutics has encouraged the continuing effort to develop a more complete understanding of the catalytic mechanism of glycosidases through the synthesis and subsequent kinetic and structural analysis of novel inhibitors. Glycosidases catalyze glycosidic bond hydrolysis via a nucleophilic substitution reaction that can result in two possible stereochemical outcomes: net retention or inversion of anomeric configuration (6Sinnott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1490) Google Scholar, 7McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar). This distinction divides glycosidases into two broad families: the retaining and the inverting glycosidases. Interestingly, when glycosidases are grouped into families based on primary amino acid sequence similarity (8Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2615) Google Scholar), this distinction holds; all enzymes within a family appear to catalyze reactions with the same stereochemical outcome (9Henrissat B. Davies G. Curr. Opin. Struct. Biol. 1997; 7: 637-644Crossref PubMed Scopus (1406) Google Scholar). There are currently more than 70 families of which at least 30 contain members whose three-dimensional structure has been determined (visit afmb.cnrs-mrs.fr/∼pedro/CAZY/db.html on the Web) (10Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1182) Google Scholar). Although glycosidases are a structurally diverse group of enzymes, two key active site carboxyl groups are conserved throughout the majority of glycosidase families and are intimately involved in glycosidic bond hydrolysis (7McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar). Inverting glycosidases are understood to use a single-displacement mechanism in which the two carboxyl groups, acting as general base and general acid catalysts, are spaced ∼10.5 Å apart, a distance large enough to allow the substrate and a water molecule to bind between them (6Sinnott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1490) Google Scholar, 7McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1607) Google Scholar). Cleavage of the glycosidic linkage involves protonation of the glycosidic oxygen by the general acid-catalyst in concert with general base-catalyzed nucleophilic attack of a water at the anomeric center. The net result is a hemiacetal product with an anomeric configuration that is inverted relative to that of the substrate. In contrast, the analogous carboxyl groups in retaining glycosidases are typically spaced 5.5 Å apart and are involved in a double-displacement mechanism (6Sinnott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1490) Google Scholar, 7McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1607) Google Scholar). The first carboxyl group promotes general acid-catalyzed cleavage of the glycosidic bond followed by nucleophilic attack at the anomeric center by the second carboxylate to form a covalent glycosyl-enzyme intermediate (see Fig. 1, lower pathway). General base-catalyzed nucleophilic attack of an incoming water molecule at the anomeric center of the intermediate yields a hemiacetal with the same anomeric configuration as the substrate. Alternatively, the configuration-retaining β-N-acetylhexosaminidases from families 18 and 20, which lack an apparent enzymic nucleophile, distort the substrate such that the carbonyl oxygen atom of the adjacent 2-acetamido group becomes appropriately positioned to act in place of the missing nucleophilic carboxylate (see Fig. 1, upper pathway) (12Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (326) Google Scholar, 13Tews I. Terwisscha van Scheltinga A.C. Perrakis A. Wilson K.S. Dijkstra B.W. J. Am. Chem. Soc. 1997; 119: 7954-7959Crossref Scopus (272) Google Scholar, 14Mark B.L. Wasney G.A. Salo T.J. Khan A.R. Cao Z. Robbins P.W. James M.N. Triggs-Raine B.L. J. Biol. Chem. 1998; 273: 19618-19624Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Drouillard S. Armand S. Davies G.J. Vorgias C.E. Henrissat B. Biochem. J. 1997; 328: 945-949Crossref PubMed Scopus (84) Google Scholar). Indeed, it has been shown that the enzymic nucleophile of the family 7 β-retaining endoglucanase I from Fusarium oxysporumoverlaps the position of the 2-acetamido carbonyl oxygen of chitobiose bound to the family 20 chitobiase from Serratia marcescens when the two structures are superimposed (16Sulzenbacher G. Driguez H. Henrissat B. Schulein M. Davies G.J. Biochemistry. 1996; 35: 15280-15287Crossref PubMed Scopus (228) Google Scholar). Anchimeric assistance by the neighboring 2-acetamido group of the substrate replaces the need for a second enzymic carboxylate and results in the formation of an enzyme-stabilized oxazolinium ion intermediate (17Knapp S. Vocadlo D. Gao Z. Kirk B. Lou J. Withers S.G. J. Am. Chem. Soc. 1996; 118: 6804-6805Crossref Scopus (226) Google Scholar, 18Mark B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The intermediate is by general base-catalyzed nucleophilic attack of water at the anomeric center in a analogous to the double-displacement mechanism B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). structures the reaction of inverting and retaining glycosidases are to have ion (7McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar, J.D. Withers S.G. Biochem. J. PubMed Scopus Google Scholar, M.N. Withers S.G. Biochemistry. 1995; PubMed Scopus Google Scholar). The azasugar inhibitors of the and isofagomine are to found within ion structures (1Heightman T.D. Vasella A.T. Angew. Chem. Int. Ed. Engl. 1999; 38: 750-770Crossref PubMed Google Scholar, M. Chem. 1998; Scopus Google Scholar, M. J. Am. Chem. Soc. 1998; Scopus Google Scholar). to be protonated and when bound in the enzyme active the endocyclic nitrogen of azasugar inhibitors with enzyme Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar). Interestingly, on the nitrogen is at the position to endocyclic oxygen as in the or at the anomeric as for the isofagomine appears to be for and β-retaining M. Chem. 1998; Scopus Google Scholar). This was to result from the position of the enzyme nucleophile within the active of these Indeed, it was in a retaining the nucleophile is positioned such that its carboxyl form a interaction with the anomeric center and endocyclic oxygen of the substrate van L. Withers S.G. Dijkstra B.W. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar). is that this interaction of the class of azasugar the protonated nitrogen replaces the endocyclic a hydrogen bond to the carbonyl oxygen of the enzyme nucleophile (1Heightman T.D. Vasella A.T. Angew. Chem. Int. Ed. Engl. 1999; 38: 750-770Crossref PubMed Google Scholar, M. J. Am. Chem. Soc. 1998; Scopus Google Scholar). β-retaining the carboxyl of the nucleophile with the substrate from the and form instead a interaction with the anomeric center and of the substrate van L. Withers S.G. Dijkstra B.W. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar). interactions with the endocyclic the enzyme nucleophile of retaining β-glycosidases with the isofagomine class of whose protonated nitrogen is at the anomeric center and is to a hydrogen bond to the enzyme nucleophile M. Chem. 1998; Scopus Google Scholar). In of this is the recent crystallographic complex of a potent isofagomine inhibitor bound to a retaining family a strong interaction between the protonated nitrogen of the inhibitor and the enzyme nucleophile Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar). with the class of which contain a adjacent to the ring nitrogen at the position by the on this This class of inhibitor bound more than the and with to and β-glycosidases H. H. A. M. J. Am. Chem. Soc. PubMed Scopus Google Scholar). in the isofagomine class of inhibit a β-retaining glycosidase that assistance and an apparent enzymic The 1-N-azasugar inhibitor (2 R,3 R,4 S,5 hydrochloride Fig. was synthesized and assayed for its ability to inhibit Streptomyces SpHEX is a β-retaining family 20 an enzymic nucleophile B.L. Wasney G.A. Salo T.J. Khan A.R. Cao Z. Robbins P.W. James M.N. Triggs-Raine B.L. J. Biol. Chem. 1998; 273: 19618-19624Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Mark B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). instead 2-acetamido group in a catalytic mechanism as The crystallographic structure of the complex of GalNAc-isofagomine bound to SpHEX a novel for the inhibitor and insight into its potent toward this family 20 The S. D. 2000; PubMed Scopus Google Scholar) Fig. was in of acid at 30 of water was and the at was for 20 and to to a The reaction was to a to in of and with of The was to to a was and with The residue was with of a The was with (2 and to 18 of GalNAc-isofagomine Fig. as a two atom for ion of GalNAc-isofagomine Fig. SpHEX was in as an and acid as B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). SpHEX was at in and was for for GalNAc-isofagomine at p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside as substrate in a reaction of and by of SpHEX the reaction determined by the in at from the of in a determined by a of the experimental the Scholar). to use in SpHEX was and and to with a of the complex by at The enzyme was with GalNAc-isofagomine for 30 and in a with and to from which within at the from a of the complex to within a to the was from the for to the its and Z. 1997; PubMed Scopus Google Scholar). The and are the and and are the and structure to GalNAc-isofagomine in the SpHEX active structure from a of SpHEX B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a in the A.T. J. M. Biol. 1998; PubMed Scopus Google Scholar), the molecular of SpHEX was positioned into the of the GalNAc-isofagomine complex followed by of from the enzyme to it into the of the GalNAc-isofagomine complex and of The to the of the bound and a of the inhibitor was into the the T.A. A. 1991; PubMed Scopus Google Scholar). and for the GalNAc-isofagomine G.J. on 1995; Scholar), and subsequent of and with a within the A.T. J. M. Biol. 1998; PubMed Scopus Google Scholar) was The are in and and for in the and of all and for in the and of all of Å in to the from is the and is the of all of Å by by and for in the and of all The in to the is the and is the of all of by J. Google Scholar). in a and structure have been into the The potency of isofagomine inhibitors for retaining β-glycosidases has been to the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the “anomeric” center of the inhibitor and the catalytic nucleophile of the enzyme Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar). family 20 glycosidases lack the enzymic nucleophile to form this strong it was that be inhibitors of family 20 glycosidases. to this GalNAc-isofagomine Fig. was found to act as a potent competitive inhibitor of the family 20 glycosidase SpHEX, a of 2.7 when as substrate into the mechanism by which GalNAc-isofagomine SpHEX has been by the structure of the complex of the complex to and the structure refined to an of and The crystallographic for the of an for GalNAc-isofagomine The inhibitor was in the of the SpHEX active site The azasugar ring of the inhibitor a that of a the endocyclic nitrogen is Å from a formed by and replaces the atom found in atom is Å on the of this within the azasugar ring also the of the ring as with the structure of N-acetyl-galactosamine Struct. Google Scholar) The ring of GalNAc-isofagomine from the for the azasugar ring of isofagomine bound to the family glycosidase from Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar), that the of GalNAc-isofagomine the to occur for and the ion of the natural substrate. The ion is to be a or with and a (6Sinnott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1490) Google Scholar). The of these the is for of the of and the at the anomeric center of the ion the of GalNAc-isofagomine is not and with the for isofagomine bound to it appears the in the isofagomine class of the ion the of the azasugar ring that of the ring of the intermediate that reactions by β-retaining glycosidases B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. D. Withers S.G. Nat. Struct. Biol. 1996; 3: PubMed Scopus Google and group for GalNAc-isofagomine and Struct. Google in for GalNAc-isofagomine and in for GalNAc-isofagomine and in for GalNAc-isofagomine and in a The SpHEX active site a of and that is for the 2-acetamido group in position for nucleophilic attack and the ion intermediate from via B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the structure of N-acetyl-galactosamine Struct. Google Scholar), which has a of the 2-acetamido group of GalNAc-isofagomine was found to be the bond when bound to SpHEX, in a of This the bond for a hydrogen bond between the carbonyl oxygen atom of the 2-acetamido group of the inhibitor and the proton of the endocyclic nitrogen This hydrogen bond is to the hydrogen bond between of isofagomine and the enzymic nucleophile of to which it is bound Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar). This the that the carbonyl oxygen atom of the 2-acetamido group in and in place of an enzymic nucleophile reactions by family 20 glycosidases B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of the crystallographic between SpHEX and GalNAc-isofagomine and B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), The refined are as the of the GalNAc-isofagomine complex are shown in and the of complex are shown in of the GalNAc-isofagomine complex are shown in the oxygen of complex are shown in of GalNAc-isofagomine and are shown in and The was in the T.A. A. 1991; PubMed Scopus Google Scholar) and as in a B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google The for GalNAc-isofagomine SpHEX are with the for the 2-acetamido group of the of chitobiose to the family 20 chitobiase from S. marcescens The molecule structure of chitobiose the carbonyl oxygen atom of the 2-acetamido group of the to be to with the Struct. Google Scholar). the complex between chitobiose and the the 2-acetamido group of the residue in the to be its bond with a of This bond in with a of the ring from a toward a the carbonyl oxygen of the 2-acetamido group of chitobiose to within Å of the anomeric it for attack at and formation of a ion intermediate (12Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (326) Google Scholar). The between GalNAc-isofagomine and chitobiose is the distance between and the carbonyl oxygen atom of the 2-acetamido group of GalNAc-isofagomine as with the distance between the analogous in chitobiose bound to the of GalNAc-isofagomine bound to SpHEX is to the ring of the intermediate a catalytic the in distance between the nucleophile and anomeric for GalNAc-isofagomine and chitobiose can be to that occur in the ring as the reaction from the complex to the The ring from a as in the complex between chitobiose and to a as for the ion intermediate B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and GalNAc-isofagomine bound to SpHEX. a catalytic SpHEX a hydrogen bond from the group of to the 2-acetamido carbonyl helping to the carbonyl oxygen with the anomeric center of the substrate B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to form the oxazolinium ion intermediate results in a on the protonated 2-acetamido nitrogen SpHEX this by a hydrogen bond from to the carboxylate of B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these interactions with and occur between SpHEX and the 2-acetamido group of and the 2-acetamido group into position within the The 2-acetamido group of GalNAc-isofagomine is not expected to a that SpHEX has to a protonated and 2-acetamido group nitrogen it is possible that the enzyme the of GalNAc-isofagomine its in a of on the carbonyl The with the on the protonated endocyclic nitrogen by the hydrogen bond in Fig. the 2-acetamido nitrogen be through of a hydrogen bond to the carboxylate of in a to the of the ion intermediate B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The hydrogen bond from the group of also the of the carbonyl oxygen of of the of the natural substrate the of the 2-acetamido carbonyl oxygen atom and the of by this family of Indeed, in the to the formation and of the oxazolinium it is that these interactions are This structure of GalNAc-isofagomine bound to SpHEX is the first with a configuration to be solved in complex with a family 20 of it has been in the complex (12Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (326) Google Scholar) and enzyme intermediate B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that family 20 glycosidases form a interaction between and of the substrate and the two of the group of a conserved residue SpHEX, This interaction appears to be for substrate for the of this conserved residue in SpHEX results in a in and a in relative to B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in that this residue is intimately involved in of the the reaction the crystallographic complex between SpHEX and GalNAc-isofagomine that the enzyme a interaction between and the configuration of GalNAc-isofagomine SpHEX and the related family 20 glycosidases catalyze the of of and A of the structures of SpHEX in complex with the intermediate or GalNAc-isofagomine that a hydrogen bond with the group of of the ring is of a or when is in an position as for the azasugar ring it is and positioned to form a hydrogen bond with the group of the complex the atom of GalNAc-isofagomine a interaction with the carboxyl group of and not with and SpHEX is conserved in family 20 that this residue is for of The carboxylate of this conserved residue has also been shown to form a hydrogen bond with of and of configuration as in the structure of SpHEX in complex with the intermediate B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and in complex with chitobiose (12Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (326) Google Scholar). the result from the crystallographic analysis of the complex between SpHEX and GalNAc-isofagomine is the 2.8-Å interaction between the general acid-base residue Glu-314 and the equatorial proton of the endocyclic nitrogen of the isofagomine inhibitor and a interaction between the protonated endocyclic nitrogen of an isofagomine inhibitor and the catalytic residue has not been in the complex of an isofagomine and retaining Withers S.G. Biochemistry. 2000; PubMed Scopus Google Scholar). The ability of GalNAc-isofagomine to form such a hydrogen bond with the general acid-base residue of a family 20 glycosidase appears to result from the ability of the catalytic nucleophile to in concert with the azasugar ring within the active site of SpHEX to the position of the intermediate bound within the active site of SpHEX B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the azasugar ring of GalNAc-isofagomine is an by and such that the endocyclic nitrogen is to Glu-314 a hydrogen bond with the carbonyl oxygen atom of the 2-acetamido group and with the SpHEX structure, Glu-314 to the of GalNAc-isofagomine with a such that is to within hydrogen distance of the endocyclic nitrogen of The in the of in with the of the distance between the endocyclic nitrogen of GalNAc-isofagomine and of Glu-314 by Å as with the distance between the analogous in the complex B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the potent of GalNAc-isofagomine toward SpHEX, this interaction appears to for the missing electrostatic interaction that between the protonated endocyclic nitrogen of an isofagomine inhibitor and a glycosidase an enzymic The unexpected potency of GalNAc-isofagomine toward SpHEX the need for molecular analysis of interactions to understand the mechanism by which a molecule the catalytic of its enzyme. and structural that the isofagomine class of inhibitors act as a competitive inhibitor of family 20 glycosidases. kinetic analysis of the of GalNAc-isofagomine toward SpHEX and it was through the analysis of the crystallographic complex formed between these two that insight into the mechanism of this unexpected was and the of for assistance with

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