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• W2041888654 abstract "Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER α-mannosidase I and subsequent recognition by the ER degradation-enhancing α-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER α-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel 3S1 sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the 1C4 conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the –1 subsite. A novel 3H4 conformation is proposed as the exploded transition state. Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER α-mannosidase I and subsequent recognition by the ER degradation-enhancing α-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER α-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel 3S1 sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the 1C4 conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the –1 subsite. A novel 3H4 conformation is proposed as the exploded transition state. Newly synthesized membrane and secretory proteins are commonly glycosylated as they enter the lumen of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; EDEM, ER degradation-enhancing α-mannosidase-like protein; ERManI, ER α-mannosidase I; dMNJ, 1-deoxymannojirimycin; SPR, surface plasmon resonance; HPLC, high performance liquid chromatography; GH, glycosylhydrolase; Man8B, an isomer of Man8GlcNAc2 where the central branch mannose residue had been removed from the standard Man9GlcNAc2 structure (see Fig. 1 of Ref. 19Tempel W. Karaveg K. Liu Z.J. Rose J. Wang B.C. Moremen K.W. J. Biol. Chem. 2004; 279: 29774-29786Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar); MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; HEPBS, N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid); RU, response unit. (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar). The protein-linked glycans are immediately trimmed, and the resulting structures subsequently serve as ligands for the luminal lectin chaperones, calnexin and calreticulin, which act in conjunction with other chaperones and thiol disulfide oxidoreductases to help facilitate protein folding, oligomerization, and disulfide bond formation (2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1630) Google Scholar). Release from the chaperones and packaging into anteriograde transport vesicles is generally accomplished only upon completion of the folding process. In contrast, proteins that fail to fold continually re-engage the chaperone machinery either until folding is achieved or until the nascent glycoproteins acquire a signal for disposal through the ER-associated degradation (ERAD) pathway (3Cabral C.M. Liu Y. Sifers R.N. Trends Biochem. Sci. 2001; 26: 619-624Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Many normal proteins, as well as many “defective” proteins in loss-of-function human genetic diseases, have delayed rather than defective folding kinetics that result in the premature disposal of the newly synthesized polypeptides (2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1630) Google Scholar). Thus, the percentage of transported functional protein is defined by a competition between the kinetics of conformational maturation versus the rate of acquiring the targeting signal for protein disposal. The present model for ERAD invokes the specific cleavage of a single mannose residue from the Man9GlcNAc2 glycan processing intermediate by the slow acting enzyme, ER mannosidase I (ERManI), as a rate-limiting initiation step for targeting incompletely folded glycoproteins for disposal (4Wu Y. Swulius M.T. Moremen K.W. Sifers R.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8229-8234Crossref PubMed Scopus (151) Google Scholar). The trimmed oligosaccharide, in the context of an unfolded polypeptide, is subsequently recognized by a collection of luminal ER lectins, termed EDEMs, which are nonhydrolytic homologs of ERManI (2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1630) Google Scholar, 5Molinari M. Calanca V. Galli C. Lucca P. Paganetti P. Science. 2003; 299: 1397-1400Crossref PubMed Scopus (390) Google Scholar, 6Oda Y. Hosokawa N. Wada I. Nagata K. Science. 2003; 299: 1394-1397Crossref PubMed Scopus (392) Google Scholar, 7Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (389) Google Scholar, 8Mast S.W. Diekman K. Davis A.W. Karaveg K. Sifers R.N. Moremen K.W. Glycobiology. 2005; (in press)PubMed Google Scholar). The nature of the complex interactions between misfolded glycoproteins, EDEMs, and other ER-associated components has still not been entirely resolved, but present models envisage a “handing off” of the ERAD substrate to the Sec61 translocon pore complex for retrotranslocation and subsequent proteasomal degradation (2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1630) Google Scholar). ERManI, EDEMs, and a collection of Golgi processing hydrolases, termed Golgi α-mannosidases IA, IB, and IC, are all members of glycosylhydrolase (GH) family 47 (9Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar, 56Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1195) Google Scholar). The hydrolytic members of this family have unique branch specificities that are required for the complete trimming to the key Man5GlcNAc2 intermediate structures necessary for maturation into complex type glycans on cell surface and secreted glycoproteins (9Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar, 10Herscovics A. Biochim. Biophys. Acta. 1999; 1426: 275-285Crossref PubMed Scopus (123) Google Scholar, 11Herscovics A. Pinto B.M. Comprehensive Natural Products Chemistry. Elsevier, New York1999: 13-35Google Scholar). Despite sequence similarities with family 47 hydrolases, EDEM family members appear to lack hydrolase activity, but they are proposed to accomplish their lectin function in ERAD by a mechanism analogous to glycan recognition during catalysis by the true hydrolases (2Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1630) Google Scholar, 5Molinari M. Calanca V. Galli C. Lucca P. Paganetti P. Science. 2003; 299: 1397-1400Crossref PubMed Scopus (390) Google Scholar, 6Oda Y. Hosokawa N. Wada I. Nagata K. Science. 2003; 299: 1394-1397Crossref PubMed Scopus (392) Google Scholar, 7Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (389) Google Scholar, 8Mast S.W. Diekman K. Davis A.W. Karaveg K. Sifers R.N. Moremen K.W. Glycobiology. 2005; (in press)PubMed Google Scholar). Thus, understanding how this family of enzymes and lectins accomplish their functions will provide insights into the rate-limiting decision between glycoprotein maturation and disposal in the secretory pathway. The two standard mechanisms for glycosidase-mediated bond hydrolysis both involve the direct protonation of the glycosidic oxygen by an enzyme-associated catalytic acid and the attack of the anomeric carbon atom by a nucleophile (12Rye C.S. Withers S.G. Curr. Opin. Chem. Biol. 2000; 4: 573-580Crossref PubMed Scopus (439) Google Scholar). Glycosidases with retaining mechanisms generally employ an enzyme-associated nucleophile, usually a Glu or Asp carboxylate side chain, resulting in the transient formation of an enzyme-linked intermediate that is subsequently cleaved by an incoming general base-activated water molecule to result in a released glycone that retains the configuration of the original glycosidic linkage. In contrast, glycosidases with inverting mechanisms employ the direct nucleophilic attack by a general base-activated water molecule to result in inversion of configuration of the released glycone. Both mechanisms require the formation of a transition state carrying a considerable positive charge delocalized between the anomeric center (C-1) and the ring oxygen (O-5) (12Rye C.S. Withers S.G. Curr. Opin. Chem. Biol. 2000; 4: 573-580Crossref PubMed Scopus (439) Google Scholar, 13Zechel D.L. Withers S.G. Curr. Opin. Chem. Biol. 2001; 5: 643-649Crossref PubMed Scopus (134) Google Scholar, 14Davies G.J. Ducros V.M. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar). The resulting partial double bond character of the C-1–O-5 bond requires co-planarity of C-5, O-5, C-1, and C-2 at or near the transition state. Among the potential pseudorotational conformational itineraries for sugar pyranose ring interconversions, there are four potential conformations where the planarity of C-5, O-5, C-1, and C-2 is satisfied (Fig. 1, 2,5B, B2,5, 4H3, and 3H4) (14Davies G.J. Ducros V.M. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar). Recent data have suggested that three of these potential transition state conformations (2,5B, B2,5, and 4H3) are employed among the known GH families (14Davies G.J. Ducros V.M. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar, 15Guimaraes B.G. Souchon H. Lytle B.L. Wu J.H.D. Alzari P.M. J. Mol. Biol. 2002; 320: 587-596Crossref PubMed Scopus (83) Google Scholar, 16Golan G. Shallom D. Teplitsky A. Zaide G. Shulami S. Baasov T. Stojanoff V. Thompson A. Shoham Y. Shoham G. J. Biol. Chem. 2004; 279: 3014-3024Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Guerin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Beguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (123) Google Scholar, 18Miyake H. Kurisu G. Kusunoki M. Nishimura S. Kitamura S. Nitta Y. Biochemistry. 2003; 42: 5574-5581Crossref PubMed Scopus (15) Google Scholar). Evidence favoring these conformations has come from the analysis of x-ray structures of target enzymes with trapped covalent intermediates using fluorinated substrate analogs (for retaining glycosidases), with enzymebound nonhydrolyzable substrate mimics, or with natural substrates in combination with active site mutants (14Davies G.J. Ducros V.M. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar, 15Guimaraes B.G. Souchon H. Lytle B.L. Wu J.H.D. Alzari P.M. J. Mol. Biol. 2002; 320: 587-596Crossref PubMed Scopus (83) Google Scholar, 16Golan G. Shallom D. Teplitsky A. Zaide G. Shulami S. Baasov T. Stojanoff V. Thompson A. Shoham Y. Shoham G. J. Biol. Chem. 2004; 279: 3014-3024Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Guerin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Beguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (123) Google Scholar, 18Miyake H. Kurisu G. Kusunoki M. Nishimura S. Kitamura S. Nitta Y. Biochemistry. 2003; 42: 5574-5581Crossref PubMed Scopus (15) Google Scholar). Of the four possible coplanar transition states, only the 3H4 conformation has not yet been identified as an intermediate in any glycosidase mechanism. Family 47 α-mannosidases from a variety of species have been extensively studied in regard to enzyme kinetics, substrate specificity, and structural analysis (9Moremen K. Ernst B. Hart G. Sinay P. Oligosaccharides in Chemistry and Biology: A Comprehensive Handbook. II. John Wiley and Sons, Inc., New York2000: 81-117Google Scholar, 10Herscovics A. Biochim. Biophys. Acta. 1999; 1426: 275-285Crossref PubMed Scopus (123) Google Scholar, 11Herscovics A. Pinto B.M. Comprehensive Natural Products Chemistry. Elsevier, New York1999: 13-35Google Scholar, 19Tempel W. Karaveg K. Liu Z.J. Rose J. Wang B.C. Moremen K.W. J. Biol. Chem. 2004; 279: 29774-29786Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar, 22Van Petegem F. Contreras H. Contreras R. Van Beeumen J. J. Mol. Biol. 2001; 312: 157-165Crossref PubMed Scopus (34) Google Scholar, 23Lobsanov Y.D. Vallee F. Imberty A. Yoshida T. Yip P. Herscovics A. Howell P.L. J. Biol. Chem. 2002; 277: 5620-5630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Mutagenesis studies of potential residues involved in Ca2+ binding and catalysis have also been carried out for S. cerevisiae ERManI (24Lipari F. Herscovics A. Biochemistry. 1999; 38: 1111-1118Crossref PubMed Scopus (38) Google Scholar), but these studies predated the structural determination of either the yeast (21Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar) or human (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) enzymes, and the roles of these residues in catalysis and substrate binding have not been revisited. Two classes of α-mannosidase co-complexes have been characterized by x-ray diffraction. Putative enzyme-glycan product co-complexes have been isolated for both ER and Golgi family 47 hydrolases, revealing the structural basis for branch specificity by these enzymes (19Tempel W. Karaveg K. Liu Z.J. Rose J. Wang B.C. Moremen K.W. J. Biol. Chem. 2004; 279: 29774-29786Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Vallee F. Lipari F. Yip P. Sleno B. Herscovics A. Howell P.L. EMBO J. 2000; 19: 581-588Crossref PubMed Scopus (86) Google Scholar). In addition, co-complexes between ER-ManI and the inhibitors, kifunensine and 1-deoxymannojirimycin (dMNJ), have revealed several unprecedented aspects of glycone binding and hydrolysis by this family of enzymes (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Both inhibitors were found to bind in the –1 subsite at the core of the enzyme (αα)7 barrel catalytic domain (Fig. 2) in the equivalent of an unusual high free energy 1C4 conformation facilitated both by a direct coordination of the inhibitor O-2′ and O-3′ hydroxyls with an enzyme-associated Ca2+ ion and by a matrix of hydrogen bonds and hydrophobic interactions. A novel inverting glycosidase mechanism was proposed where the water nucleophile was one of the ligands directly coordinated with the enzyme-bound Ca2+ ion, and the catalytic acid was proposed to act indirectly in a through-water protonation of the glycosidic oxygen (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The unusual nature of this proposed mechanism precluded an unambiguous identification of the catalytic acid and base residues. We describe here the kinetics and binding characteristics of potential catalytic mutants as well as a co-complex of ERManI with a nonhydrolyzable thiodisaccharide pseudosubstrate. These data demonstrate the roles of various active site residues as well as the novel conformational itinerary during glycoside bond hydrolysis. Synthesis of Methyl-2-S-(α-d-mannopyranosyl)-2-thio-α-d-mannopyranoside—The previous reported synthesis of the thiodisaccharide, methyl-2-S-(α-d-mannopyranosyl)-2-thio-α-d-mannopyranoside, was achieved using the coupling reaction of methyl 4,6-O-benzylidene-2-O-trifluoromethanesulfonyl-α-d-mannopyranoside with the thiolate anion of 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranose (25Johnston B.D. Pinto B.M. Carbohydr. Res. 1998; 310: 17-25Crossref Scopus (19) Google Scholar). We chose an alternate strategy using the alternative key coupling reaction of methyl 3-O-benzyl-4,6-O-benzylidene-2-S-thioacetyl-α-d-mannopyranoside with 2,3,4,6-tetra-O-acetyl-d-mannopyranosyl bromide and characterized the synthetic product by NMR as described in the Supplementary Data. Mutagenesis, Expression, and Purification of Human ERManI—The expression and purification of the human ERManI catalytic domain has been described previously (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 26Gonzalez D.S. Karaveg K. Vandersall-Nairn A.S. Lal A. Moremen K.W. J. Biol. Chem. 1999; 274: 21375-21386Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The cDNA encoding ERManI in the pPICZαA vector (Invitrogen) was used to perform site-directed mutagenesis using the QuikChange™ mutagenesis kit from Stratagene (La Jolla, CA) based on the sequence of human ERManI (GenBank™ accession number AF145732). The full coding region of each mutant was sequenced to confirm that only the desired mutation was generated. The plasmid constructs were then used to transform the Pichia pastoris strain X-33, and zeocin-resistant colonies were screened for ERManI expression by performing Western blots using conditioned medium from induced cultures as previously described (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). ERManI was detected using a rabbit polyclonal anti-human ERManI antibody generated to the recombinant wild type enzyme, and the antibody was detected using a peroxidase-conjugated anti-rabbit IgG secondary antibody. Mutant enzymes were expressed in 1-liter shake flask cultures by induction in BMMY media, and the enzyme was purified from the conditioned media as previously described (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Man9GlcNAc2-PA and Glycopeptide Preparation—Crude soybean agglutinin was extracted from soy flour by acid and ammonium sulfate precipitation (27Evers D.L. Hung R.L. Thomas V.H. Rice K.G. Anal. Biochem. 1998; 265: 313-316Crossref PubMed Scopus (25) Google Scholar) and was subsequently denatured, reduced, and carboxyamidomethylated in 8 m guanidine HCl containing 50 mm dithiothreitol and 100 mm iodoacetamide (28Rice K.G. Rao N.B. Lee Y.C. Anal. Biochem. 1990; 184: 249-258Crossref PubMed Scopus (43) Google Scholar) prior to proceeding to elastase digestion (29Deras I.L. Sano M. Kato I. Lee Y.C. Anal. Biochem. 2000; 278: 213-220Crossref PubMed Scopus (7) Google Scholar). The Man9GlcNAc2-glycopeptide was recovered by affinity chromatography using concanavalin A-Sepharose and further purified by HPLC on a Cosmosil C18 column (30Deras I.L. Kawasaki N. Lee Y.C. Carbohydr. Res. 1998; 306: 469-471Crossref PubMed Scopus (7) Google Scholar) for SPR studies. For enzyme assays, Man9GlcNAc2 was liberated from the peptide by peptide:N-glycosidase F digestion (31Sutton C.W. O'Neill J.A. Methods Mol. Biol. 1997; 64: 73-79PubMed Google Scholar) and derivatized with pyridylamine (32Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar, 33Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1929) Google Scholar). Enzyme Assays—The purified wild type and mutant enzymes were assayed for α1,2-mannosidase activity using pyridylamine-tagged Man9GlcNAc2 (Man9GlcNAc2-PA) as substrate (32Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar). The enzyme reactions (20 μl) were carried out in 96-well plates by adding 10 μl of enzyme in 300 mm NaCl and 10 mm CaCl2 to a mixture of 5 μl of 4× universal buffer (80 mm succinic acid, 80 mm MES, 80 mm HEPBS, 80 mm HEPES, and 80 mm CHES adjusted to pH with 5 m NaOH) and 5 μl of substrate. The reactions were performed at 37 °C for the indicated times and stopped by the addition of 20 μl of 1.25 m Tris-HCl (pH 7.6) to the reaction mixture. The enzymatic products were resolved and quantitated using a Hypersil APS-2 NH2-HPLC column (32Lal A. Pang P. Kalelkar S. Romero P.A. Herscovics A. Moremen K.W. Glycobiology. 1998; 8: 981-995Crossref PubMed Scopus (104) Google Scholar). Initial rates (v) for the enzymes were determined at various substrate concentrations ranging from 10 to 300 μm. The catalytic coefficient (kcat) and Michaelis constant (Km) values were determined by fitting initial rates to a Michaelis-Menten function by nonlinear regression analysis using SigmaPlot (Jandel Scientific, San Rafael, CA)). kcat/Km values were derived from reciprocal plots of v and [S] where needed. pH Rate Dependence Analysis—Values for kcat and Km were determined from initial rates of enzyme reactions in the pH range from 4 to 10 using the 1× universal buffer described above. Plots of log(kcat/Km) versus pH were used to estimate values of the macroscopic enzyme ionization constants (pKE1 and pKE2) by linear extrapolation of the acidic and basic limbs of the curve (Fig. 3). Binding Studies by Surface Plasmon Resonance (SPR)—SPR analyses were conducted using a Biacore 3000 apparatus (Biacore AB, Piscataway, NJ). Recombinant ERManI was immobilized on the SPR chip surfaces at 25 °C by the amine coupling method (34O'Shannessy D.J. Brigham-Burke M. Peck K. Anal. Biochem. 1992; 205: 132-136Crossref PubMed Scopus (227) Google Scholar). The flow cells were activated by injecting a mixture of 50 mmN-hydroxysuccinimide and 200 mm 1-ethyl-3-(dimethylaminopropyl)carbodiimide over the CM5 sensor chip surface for 7 min at 5 μl/min. The recombinant proteins (30 μg/ml) prepared in 10 mm sodium succinic acid (pH 6.0) were passed through a 0.2-μm polyvinylidene difluoride filter (Millipore Corp.) and diluted in the same buffer to obtain a concentration of 5 μg/ml prior to injection onto the activated surface. The desired immobilization level was achieved by specific contact time. The remaining reactive groups were blocked by injection of 1 m ethanolamine-HCl at pH 8.5 for 7 min at 5 μl/min. The immobilization efficiency for ERManI was about 2500 RU/min at a flow rate of 5 μl/min in HPB-EP buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.01% polysorbate P20) at 25 °C. Mock derivatized flow cells served as reference surfaces. The binding analyses were performed at 10 °C with a continuous flow (30 μl/min) of running buffer (10 mm MES, pH 7.0, 300 mm NaCl, and 5 mm CaCl2). Analytes were prepared in running buffer by 2-fold serial dilution to obtain an appropriate concentration range. A concentration series of Man9GlcNAc2-glycopeptide (0.4–400 μm) was analyzed over a low density immobilization surface of recombinant protein (3000 RU), whereas disaccharides (16–1000 μm) and dMNJ (2–1000 μm) were analyzed over a high density immobilization surface of recombinant protein (10,000 RU). Samples were injected for 1 min followed by at least 3 min of dissociation phase. The base line returned to the original response level in 5 min for all analytes described here without a further regeneration of the chip surface. SPR data for each concentration of analyte were collected in duplicate and globally fit to a 1:1 Langmuir binding algorithm model to calculate the on-rate (ka), the off-rate (kd), and the equilibrium dissociation constant (kd/ka = KD) using Biaevaluation version 3.1 software (35Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (656) Google Scholar). Alternatively, the maximal equilibrium sensorgram values were used to plot a saturation binding curve and calculate values for the equilibrium dissociation constant (KD) directly. Crystallization and X-ray Diffraction—In an effort to obtain higher resolution crystal structure data than was previously obtained for human ERManI (20Vallee F. Karaveg K. Herscovics A. Moremen K.W. Howell P.L. J. Biol. Chem. 2000; 275: 41287-41298Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), a sparse matrix screen (36Jancarik J. Kim S-H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar) was performed with the Crystal Screen HT™ from Hampton Research, carried out using an Oryx 6 crystallization robot (Douglas Instruments) in Nunc HLA plates. The crystallization drop was prepared by mixing equal volumes (0.5 μl) of the protein and the screening solutions (final protein concentration 10 mg/ml) and sealed with paraffin oil (15 μl). The plate was then overlaid with a mixture of silicone and paraffin oils (30:70) (37Chayen N.E. Shaw Stewart P.D. Maeder D.L. Blow D.M. J. Appl. Crystallogr. 1990; 23: 297-302Crossref Google Scholar, 38D'Arcy A. Elmore C. Stihle M. Johnston J.E. J. Cryst. Growth. 1996; 168: 175-180Crossref Scopus (79) Google Scholar) and kept at 18 °C. Crystal formation was monitored after 3 days. Additive screening was carried out by mixing equal volumes (0.5 μl) of the protein solution, the optimized crystallization solution (24% (w/v) polyethylene glycol 4000, 100 mm MES/NaOH, pH 6.0, and 50 mm ammonium sulfate), and 3× additive screen components (Hampton Research, catalog number HR2-428). The addition of 1,4-butanediol was found to produce the best crystals and improved diffraction to beyond 1.35 Å for the native enzyme (data not shown). Since we had a limited supply of the thiodisaccharide substrate analogue, direct co-crystallization was attempted using the above conditions without further optimization. For co-crystallization, the thiodisaccharide (100 mm in H2O) was added to the protein solution to yield 10 mg/ml recombinant protein, 20 mm MES, pH 7.0, 150 mm NaCl, 5 mm CaCl2, 750 mm NDSB 201, and 50 mm thiodisaccharide and incubated for 2 h at room temperature prior to crystallization. A hanging drop was prepared over 1 ml of mother liquor containing 24% (w/v) polyethylene glycol 4000, 100 mm MES/NaOH, pH 6.0, 50 mm ammonium sulfate, and 10% (v/v) 1,4-butanediol. The crystallization drop contained 1.5 μl of protein/ligand solution and 1 μl of mother liquor. Crystals formed within 4 h at 25 °C and were mounted and flash-frozen 1 day later using 10% glycerol as cryoprotectant. Although the single co-crystals grown by this method were considerably smaller in size than that of the native crystals, one of the crystals produced diffraction data that could be scaled to 1.41 Å with good statistics and completeness (Table III).Table IIIData collection and refinement statisticsParameterValueaValues in parentheses refer to data from the high resolution shell.Data resolution (Å)48.80 to 1.41 (1.45 to 1.41)Unique observed reflections84,600 (8,225)Completeness (%)95.4 (92.6)〈I〉/〈(σI)〉41.8 (13.0)Average redundancy4.0 (3.5)Space groupP1Unit cell lengths a, b, c (Å)50.7, 53.9, 56.2Unit cell angles α, β, γ (degrees)89.5, 63.6, 62.6Matthews coefficient (Å3·Da-1)2.4Solvent content (%, v/w)47Mosaicity (degrees)0.1-0.3No. of unique reflections used in refinement76,092 (5,417)No. of reflection in free set8,507 (631)Rcryst (%)14.4 (15.8)Rfree (%)16.2 (19.3)No. of nonhydrogen atoms refined4,097Root mean square deviation (Å) bonds0.014Root mean square deviation (degrees) angles1.34Mean temperature factor (Å2)8.4Wilson temperature factor (Å2)9.3a Values in parentheses refer to data from the high resolution shell. Open table in a new tab Crystals were examined using an ACTOR/Director automated crystal-screening system (Rigaku/MSC) consisting of an FR-D high intensity rotating anode, a Max-Flux confocal optics, an AFC-9 four-axis goniometer, and a Saturn 92 CCD detector. Complete diffraction data were collected at the Advanced Photon Source, beam line 22ID, using a MARCCD-225 dete" @default.
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• W2041888654 date "2005-04-01" @default.
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• W2041888654 title "Mechanism of Class 1 (Glycosylhydrolase Family 47) α-Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control" @default.
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• W2041888654 doi "https://doi.org/10.1074/jbc.m500119200" @default.
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