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An activity-based isotope-coded affinity tagging (AB-ICAT) strategy for proteome-wide quantitation of active retaining endoglycosidases has been developed. Two pairs of biotinylated, cleavable, AB-ICAT reagents (light H(8) and heavy D(8)) have been synthesized, one incorporating a recognition element for cellulases and the other incorporating a recognition element for xylanases. The accuracy of the AB-ICAT methodology in quantifying relative glycosidase expression/activity levels in any two samples of interest has been verified using several pairs of model enzyme mixtures where one or more enzyme amounts and/or activities were varied. The methodology has been applied to the biomass-degrading secretomes of the soil bacterium, Cellulomonas fimi, under induction by different polyglycan growth substrates to obtain a quantitative profile of the relative expression/activity levels of individual active retaining endoglycanases per C. fimi cell. Such biological profiles are valuable in understanding the strategies employed by biomass-degrading organisms in exploiting environments containing different biomass polysaccharides. This is the first report on the application of an activity-based ICAT method to a biological system.

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining β-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining β-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi β-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank™ accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ∼70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.

Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.

The inclusion of both beta-D-xylosidases and alpha-L-iduronidases within the same sequence-related family (family 39), despite the considerable difference in substrate structures and poor sequence conservation around the putative nucleophile, raises concerns about whether a common mechanism is followed by the two enzymes. A novel anchimeric assistance mechanism for iduronidases involving a lactone intermediate is one possibility. NMR analysis of the methanolysis reaction catalyzed by human alpha-L-iduronidase reveals that, as with the beta-D-xylosidases, alpha-L-iduronidase is a retaining glycosidase. Using two different mechanism-based inactivators, 5-fluoro-alpha-L-iduronyl fluoride and 2-deoxy-2-fluoro-alpha-L-iduronyl fluoride, the active site nucleophile in the human alpha-L-iduronidase was identified as Glu299 within the (295)IYNDEAD(301) sequence. The equivalent, though loosely predicted, glutamic acid was identified as the nucleophile in the family 39 beta-D-xylosidase from Bacillus sp. [Vocadlo, D., et al. (1998) Biochem. J. 335, 449-455]; thus, a common mechanism involving a covalent glycosyl-enzyme intermediate that adopts the rather uncommon (2,5)B conformation is predicted.

The formation of yeast cytochrome c peroxidase (CcP) compound I has been recognized for many years to be associated with formation of two protein-centered radicals. One of these radical sites is located at Trp191 and is directly involved in catalytic oxidation of ferrocytochrome c (Sivaraja, M., Goodin, D. B., Smith, M., Hoffman, B. M. (1989) Science 245, 738-740). The second radical has been proposed to arise from one or more tyrosyl residues of CcP. However, the tyrosyl residue (or residues) capable of forming this radical has not been identified, and the functional role of this radical remains poorly understood. In the present work, this issue has been addressed through the combined use of the spin-trapping reagent 2-methyl-2-nitrosopropane and peptide mapping by electrospray mass spectrometry to identify Tyr39 and Tyr153 as two tyrosyl residues that are capable of forming radical centers upon reaction of CcP with hydrogen peroxide. The implications of this observation to the catalytic mechanism of CcP are addressed with reference to the three-dimensional structure of CcP.

Synthesis of the potential mechanism-based inactivator of β-D-glucuronidases (5-fluoro-β-D-glucopyranosyluronic acid fluoride) was accomplished via a six-step process from D-glucuronic acid that involved radical bromination at C-5 and displacement of the bromide by fluoride. A key step in this process was the masking of the carboxylic acid as a phenacyl ester. This group is uniquely stable to conditions of photobromination and fluoride displacement, yet removable under very mild conditions. Incubation of the Escherichia coli β-glucuronidase with 5-fluoro-β-D-glucopyranosyluronic acid fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 5-fluoro-α-D-glucopyranosyluronic acid-enzyme. Peptic digestion of the 5-fluoro-α-D-glucopyranosyluronic acid-enzyme intermediate and subsequent analysis by liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer indicated the presence of a 5-fluoro-α-D-glucopyranosyluronic acid-modified peptide. This peptide was partially purified by HPLC and its sequence determined by tandem mass spectrometry in the daughter ion scan mode, permitting the identification of Glu504 as the catalytic nucleophile within the sequence ITEYGVD. This new reagent is therefore useful for the specific, mechanism-based inactivation of glycuronidases and has good potential in other studies of enzymes of this general class.Key words: β-glucuronidase, catalytic nucleophile, 5-fluoro-β-D-glucopyranosyluronic acid fluoride, electrospray MS.

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside (1) and 2', 4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett. 435, 225-228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem. 267, 18744-18752].

5-Fluoro-alpha-D-galactopyranosyl fluoride was synthesized and its interaction with the active site of an alpha-galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding (Ki = 600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies.

Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glc beta(1-6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase.

Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.

The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosykl fluoride has been synthesized, and the kinetic parameters K(M) = 0.23 mM and K(CAT)= 0.55 min(-1) for its hydrolysis by vibrio furnisi beta-N-acetylglucosaminidase (ExoII) HAVE been determined. Investigation of mixtures of enzyme with this slow substrate by electrospray mass spectrometry revealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro-beta-L-idopyranosyl-enzyme, indicating that the hydrolytic mechanism of ExoII involves the formation and rate-determining hydrolysis of a glycosyl-enzyme intermediate. Analysis of a peptic digest of the glycosyl-enzyme by HPLC/ESMS/MS in the netural-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide, in conjuction with multiple sequence alignmentsS of family 3 members, allowed the identification of ASP242 as the catalytic nucleophile within the sequence IVFSDDLSM.

Human beta-glucuronidase is a member of the Family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. The enzyme is shown to catalyze glycoside bond hydrolysis with net retention of anomeric configuration, presumably via a mechanism involving a covalent glucuronyl-enzyme intermediate. Incubation of human beta-glucuronidase with 2-deoxy-2-fluoro-beta-D-glucuronyl fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme, as observed by electrospray mass spectrometry. Regeneration of the free enzyme by hydrolysis or transglycosylation and removal of excess inactivator demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme intermediate and subsequent analysis by liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry indicated the presence of a 2-deoxy-2-fluoro-alpha-D-glucuronyl peptide. Sequence determination of the labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Glu-540 as the catalytic nucleophile within the sequence SEYGAET.

The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35.

The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 β-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.

1-aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia. In an early study of this unusual C(alpha)-C(beta) ring cleavage reaction, 1-amino-2-methylenecyclopropane-1-carboxylic acid (2-methylene-ACC) was shown to be an irreversible inhibitor of ACC deaminase. The sole turnover product was identified as 3-methyl-2-oxobutenoic acid. These results provided strong evidence supporting the ring cleavage of ACC via a nucleophilic addition initiated process, thus establishing an unprecedented mechanism of coenzyme B(6) dependent catalysis. To gain further insight into this inactivation, tritiated 2-methylene-ACC was prepared and used to trap the critical enzyme nucleophiles. Our results revealed that inactivation resulted in the modification of an active site residue, Ser-78. However, an additional 5 equiv of inhibitor was also found to be incorporated into the inactivated enzyme after prolonged incubation. In addition to Ser-78, other nucleophilic residues modified include Lys-26, Cys-41, Cys-162, and Lys-245. The location of the remaining unidentified nucleophile has been narrowed down to be one of the residues between 150 and 180. Labeling at sites outside of the active site is not enzyme catalyzed and may be a consequence of the inherent reactivity of 2-methylene-ACC. Further experiments showed that Ser-78 is responsible for abstracting the alpha-H from d-vinylglycine and may serve as the base to remove the beta-H in the catalysis of ACC. However, it is also likely that Ser-78 serves as the active site nucleophile that attacks the cyclopropane ring and initiates the fragmentation of ACC, while the conserved Lys-51 is the base required for beta-H abstraction. Clearly, the cleavage of ACC to alpha-ketobutyrate by ACC deaminase represents an intriguing conversion beyond the common scope entailed by coenzyme B(6) dependent catalysts.

The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C1 14A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glul 15 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glul 15 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu 115 and Glu 135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.

International audience; The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35.

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 Å resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid–base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032–16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2´,4´-dinitrophenyl 2-deoxy-2-fluoro-β-d-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact = 0.29±0.02 min-1 and Kinact = 0.72±0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki = 9±1 mM), a behaviour that was also observed with the substrate 2´,4´-dinitrophenyl β-d-cellobioside (kcat = 40±1 s-1, Km = 0.35±0.03 mM, Ki = 24±4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O = 0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact = 0.024 min-1, Kreact = 54 mM). Digestion of the labelled enzyme by pepsin followed by LC–ESI–tandem MS (MS–MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI–MS–MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid–base catalyst Glu-203 are conserved in all known family-12 sequences.

Thermoanaerobacterium saccharolyticum beta-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining beta-d-xylosidases and alpha-l-iduronidases. T. saccharolyticum beta-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl-enzyme intermediate. Incubation of T. saccharolyticum beta-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki=0.089 min-1, dissociation constant for the inactivator Ki=65 microM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-alpha-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.

Mannosidases play a key role in the processing of glycoproteins and thus are of considerable pharmaceutical interest and indeed have emerged as targets for the development of anti-cancer therapies. Access to useful quantities of the mammalian enzymes has not yet been achieved; therefore, jack bean mannosidase, a readily available enzyme, has become the model system. However, the relevance of this enzyme has not been demonstrated, nor is anything known about the active site structure of this, or any other, mannosidase. Hydrolysis by this enzyme occurs with net retention of sugar anomeric configuration; thus, a double displacement mechanism involving a mannosyl-enzyme intermediate is presumably involved. Two new mechanism-based inhibitors, 5-fluoro-alpha-D-mannosyl fluoride and 5-fluoro-beta-L-gulosyl fluoride, which function by the steady state trapping of such an intermediate, have been synthesized and tested. Both show high affinity for jack bean alpha-mannosidase (Ki' = 71 and 86 microM, respectively), and the latter has been used to label the active site nucleophile. The labeled peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z 1180.5 in the labeled sample. The label was cleaved from the peptide by treatment with ammonia, and the resultant unlabeled peptide was purified and sequenced by Edman degradation. The peptide identified contained only one candidate for the catalytic nucleophile, an aspartic acid. This residue was contained within the sequence Gly-Trp-Gln-Ile-Asp-Pro-Phe-Gly-His-Ser, which showed excellent sequence similarity with regions in mammalian lysosomal and Golgi alpha-mannosidase sequences. These mammalian alpha-mannosidases belong to family 38 (or class II alpha-mannosidases) in which the Asp in the above sequence is totally conserved. This finding therefore assigns jack bean alpha-mannosidase to family 38, validating it as a model for other pharmaceutically interesting enzymes and thereby identifying the catalytic nucleophile within this family.

Sweet almond beta-glucosidase is a well studied glycosidase, having been subjected to numerous kinetic analyses and inhibition studies. However, it is not known to which glycosidase family it belongs, nor is the identity of the active site nucleophile known with certainty. It can be inactivated using the specific, mechanism-based enzyme inactivator 2-deoxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by forming a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate. The glycosylated peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z = 1041 in the labeled sample. The sequence of this peptide was determined to be Ile-Thr-Glu-Gln-Gly-Val-Asp-Glu by further tandem mass spectrometric analysis in the daughter ion scan mode in conjunction with Edman degradation of the purified peptide. The identity of the labeled side chain was determined by further tandem mass spectrometric analysis in the daughter ion scan mode of a partially purified sample of the labeled peptide subjected to methyl esterification, the fragmentation pattern being consistent only with the first Glu in the sequence being labeled. The sequence around this residue is identical to that surrounding the catalytic nucleophile in many members of glycosidase Family 1, confirming the assignment of this enzyme to that family. The residue labeled is, however, different from that (Asp) identified previously in the enzyme from bitter almonds by use of conduritol epoxide affinity labels, although apparently close in the primary sequence.

Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl alpha-fluoride (4DG3 alpha F). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3 alpha F was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (k(cat) = 2 s(-1)) compared with the parent glycosyl fluoride, maltotriosyl alpha-fluoride (k(cat) = 275 s(-1)). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3 alpha F was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (k(cat) = 0.6 s(-1)). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.

The mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.

Bovine kidney lysosomal alpha-mannosidase is a family 38 alpha-mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-beta-L-gulosyl fluoride, was used to trap a glycosyl-enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis. Taking into consideration sequence alignments of this region with those of other alpha-mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.