Namrata
Jain
ab,
Mohamed A.
Attia
ab,
Wendy A.
Offen
c,
Gideon J.
Davies
c and
Harry
Brumer
*abd
aMichael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada. E-mail: brumer@msl.ubc.ca; Fax: (+1) 6048222114; Tel: (+1) 6048273738
bDepartment of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
cDepartment of Chemistry, University of York, Heslington, York YO10 5DD, UK
dDepartment of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada
First published on 24th October 2018
Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure–function analyses of XyG-specific endo-glucanases, we have synthesized a 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-D-Glcp. Key steps in the chemo-enzymatic synthesis included selective enzyme hydrolysis of XyG polysaccharide to produce the core heptasaccharide, per-O-acetylation, α-bromination, reductive glycal formation, electrophilic fluorination, SNAr glycosylation, and Zemplen deprotection. The resulting compound, XXXG(2F)-β-DNP, specifically labelled the active sites of several endo-(xylo)glucanases by accumulation of a covalent glycosyl-enzyme intermediate, as revealed by intact protein mass spectrometry. Crystallography of a complex with a Cellvibrio japonicus Glycoside Hydrolase Family 5 (GH5) endo-xyloglucanase corroborated the covalent nature of the intermediate, and further revealed the anticipated specificity for the catalytic nucleophile of this anomeric-configuration-retaining glycosidase. This specificity complements that of an analogous XXXG N-bromoacetylglycosylamine inhibitor, which labelled the catalytic acid–base sidechain in the same enzyme [Attia, et al., Biotechnol. Biofuels, 2018, 11, 45]. We anticipate that these inhibitors may find continued use in mechanistic analyses of endo-(xylo)glucanases from diverse GH families.
The use of small molecule glycomimetic inhibitors has been central to GH structure–function analyses in glycobiology.9–11 In particular, irreversible, covalent inhibitors of GHs have been extensively used to identify key substrate-binding and catalytic residues through analytical biochemistry and enzyme crystallography, and to screen for new GHs. A wide array of generally reactive, photo-activatable, or mechanism-based inhibitors based on bespoke glycan specificity motifs has been deployed, including carbasugar-epoxides (e.g. conduritol-β-epoxide, cyclophellitol), cyclopropylcarbasugars, epoxyalkyl glycosides, N-bromoacetylglycosylamines, bromoketone C-glycosides, glucosylthio-hydroquinones, aziridines, cyclosulfates, glycosylmethyl triazenes, activated phenylmethyl glycosides, various photoaffinity labels, and glycosides fluorinated at the 2- or 5-position (see comprehensive reviews and references therein,12–14 and recent primary literature15–19).
Since their introduction by Withers and coworkers 30 years ago,20 2-deoxy-2-fluoroglycosides bearing activated aglycones (and the related 2-deoxy-2,2-difluoroglycosides21–23) have been widely used for mechanistic and structural studies of diverse GHs.14 By virtue of their mechanism-based inhibition and conservative steric substitution, this class of compounds has been exceptionally useful for identifying the catalytic nucleophile by protein mass spectrometry and/or crystallography in GH families that utilize a double-displacement, anomeric-configuration-retaining mechanism (Scheme 1a).24 During catalysis, the presence of the fluorine group at C-2 destabilizes the transition state of both chemical steps, while the incorporation of a good nucleofuge, such as fluorine or 2,4-dinitrophenol (2,4-DNP), increases the rate of leaving group departure sufficiently to enable the accumulation of the 2-deoxyfluoroglycosyl-enzyme intermediate. For some GHs, inhibition is essentially complete, while in others, these inhibitors act as “slow substrates” due to demonstrable turnover to release the free enzyme,25 especially in the presence of sugars as alternate glycosyl acceptor substrates.20,21,26–31
The xyloglucans (XyGs) comprise a family of complex heteropolysaccharides, whose members are ubiquitous in land plants,32 where they can constitute up to one-quarter of the dry weight of the primary cell wall.33 A central structural feature of XyGs is a linear β(1,4)-glucan backbone that is regularly substituted with α(1,6)-xylosyl residues, which can be further extended by various other saccharide residues depending on the source tissue.34 In dicot XyGs, the core repeating unit comprises a heptasaccharide motif (Xyl3Glc4), in which three of four contiguous backbone glucosyl units are branched (Fig. 1). Consequently, such XyGs are referred to as “XXXG-type”35 in the standard shorthand, in which “G” represents an unbranched β(1,4)-linked backbone glucosyl residue, and “X” represents the disaccharide motif comprising a β(1,4)-linked backbone glucosyl residue bearing an α(1,6)-linked xylosyl sidechain.36
Fig. 1 Structure of dicot xyloglucan. Substructure nomenclature is according to Tuomivaara et al.36 and symbols are according to the Consortium for Functional Glycomics.65 |
Inspired by the success of 2-deoxy-2-fluoroglycosides as mechanism-base inhibitors and motivated by a long-standing interest in the enzymology of xyloglucan metabolism by plants and microorganisms,37–40 we present here the chemo-enzymatic synthesis of XXXG(2F)-β-DNP as a specific mechanism-based inhibitor of endo-(xylo)glucanases (EC 3.2.1.151). In particular, we demonstrate the application of this compound for the covalent labelling of exemplar configuration-retaining endo-(xylo)glucanases by protein mass spectrometry and crystallography.
TLC was performed using aluminum sheet TLC plates (0.25 mm) pre-coated with Merck silica gel 60 F254, using ethyl acetate:hexanes or water:isopropanol:ethyl acetate as solvent systems (particular solvent ratios specified below), and visualized by a UV lamp and/or 10% sulfuric acid in water with charring using a heat gun. Flash chromatography was performed using Merck silica gel 60 with ethyl acetate:hexanes or water:isopropanol:ethyl acetate as mobile phases. Fractions were analyzed by TLC, and those with the desired compounds were pooled together and evaporated under reduced pressure.
All 19F-, 13C- and 1H-NMR data were collected on a Bruker Avance 400 MHz spectrometer at room temperature (100.6 MHz and 376.5 MHz for 13C- and 19F-, respectively). The NMR spectra were referenced to solvent as follows: HOD = 4.79 ppm, CHCl3 = 7.27 ppm, 13CHCl3 = 71 ppm, C19FCl3 = 0 ppm. MALDI-MS data were collected on a Bruker Autoflex instrument in reflectron mode over m/z 700–3500 using 6-Aza-2-thiothymine (ATT) as the matrix. HRMS data were obtained using either a Waters Xevo G2-S Q-ToF or Waters/Micromass LCT ToF mass spectrometer in positive-ion mode, via direct infusion through an electrospray ion source.
The synthesis of per-O-acetylated XXXG glycal (2) was performed by adapting the method of Xu et al.42 (per-OAc)XXXG α-bromide (1, 1 g) was dissolved in 30 ml in acetic acid. A solution of Zn (31 eq.), NaOAc (18.5 eq.), and CuSO4·5H2O (0.2 eq.) was suspended in 20 mL of water and stirred for 5 min. The solution of 1 in acetic acid was slowly added to the suspension. The reaction was stirred for 3 h at room temperature, and then filtered through a Celite pad. The solvent was concentrated under reduced pressure, re-dissolved in CH2Cl2 and washed with NaHCO3 (3×) and brine (1×). The organic layer was concentrated, and flash chromatography (mobile phase ethyl acetate/hexanes 2:1, Rf = 0.3 in the same solvent mixture) followed by evaporation of the solvent under reduced pressure was used to isolate a white solid (0.63 g, 68% yield). 1H-NMR (Fig. S1,† 400 MHz, CDCl3): δ 6.42 (d, J = 6.14 Hz, 1H, H1), 5.42–5.29 (m, 7H), 5.20–3.63 (m), 1.98–2.03 (m, 54H, COCH3). 13C-NMR (Fig. S2,† 100.6 MHz, CDCl3): δ 170.76, 170.60, 170.41, 170.36, 170.34, 170.24, 170.17, 170.12, 170.09, 170.01, 169.97, 169.88, 169.85, 169.71, 169.63, 169.45, 168.97, 168.68, 168.64 (18 × CO-Ac), 145.58 (C1), 101.07, 100.72, 100.57, 98.24, 97.55, 97.17, 95.94 (C2, 6 × C1), 75.66, 75.36, 74.85, 74.78, 74.45, 74.09, 73.42, 7.38, 72.94, 72.29, 72.06, 71.90, 71.80, 71.70, 70.92, 70.77, 70.49, 70.44, 69.54, 69.49, 69.36, 69.33, 69.23, 69.18, 69.10, 67.45, 67.17 (6 × C2, 7 × C3, 7 × C4, 7 × C5), 65.87, 61.77, 59.22, 58.85 (4 × C6), 20.58–21.01 (18 × CH3). Monoisotopic m/z calculated for C75H100O49K+: 1823.4975; MALDI-ToF MS found: 1823.3. m/z calculated for C75H100O49Na+: 1807.5236; ESI-HRMS found: 1807.5282.
The synthesis of 1-hydroxy-2-deoxy-2-fluoro-(per-OAc)XXXG (3) was adapted from previously reported methods.43,44 Compound 2 (0.63 g) was dissolved in dry nitromethane (20 ml) and 1.2 eq. of Selectfluor™ was added to the solution to create a suspension, which was stirred at room temperature for 6 h, at which time water (2.4 eq.) was added. The solution was then heated to 60 °C and stirred at that temperature for 16 h. The reaction was filtered over Celite to remove the Selectfluor™ by-product and the filtrate was concentrated by rotary evaporation. Elution of the crude product through a silica column (ethyl acetate/hexanes, 3.5:1) to remove polar impurities and remaining by-products yielded a mixture of anomers (yield 0.24 g, 37%), which was used directly in the next step without further separation. 19F-NMR (Fig. S3,† 376.5 MHz, CDCl3) gluco β-anomer: −197.91 (ddd, JH2–F2 = 51.1 Hz, JH3–F2 = 13.7 Hz, JH1–F2 = 1.6 Hz, F2), gluco α-anomer: −199.30 (dd, JH2–F2 = 49.5 Hz, JH3–F2 = 12.0 Hz, JH1–F2 = 0 Hz, F2), consistent with values for the corresponding cellobioside.21,45 Monoisotopic m/z calculated for C75H101FO50Na+: 1843.5248; MALDI-ToF MS found: 1843.3; ESI-HRMS found: 1843.5494.
The synthesis of the per-O-acetylated 2′4′-dinitrophenyl β-glycoside of 2-deoxy-2-fluoro-XXXG (4) was adapted from a previously reported glycosylation method.21 A solution of 2-fluoro-dinitrobenzene (2FDNB, 2 eq.) was dissolved in dry dimethylformamide (DMF) and stirred over activated 4 Å molecular sieves overnight. Crude 3 (0.24 g) was dissolved in dry DMF (10 mL) and under dry conditions 5 eq. of 1,4-diazabicyclo[2.2.2]octane (DABCO) were added to the solution as a solid powder. The reaction was stirred for 15 min, after which it was added to the solution of 2FDNB through a syringe with an oven-dried steel needle. The reaction was stirred for 3.5 h, after which time the molecular sieves were filtered away and the DMF in the filtrate was evaporated under reduced pressure. The solution was re-dissolved in CH2Cl2 and washed with NaHCO3 (3×) and brine (1×). Flash chromatography (mobile phase ethyl acetate/hexanes 2.5:1) was used to isolated the pure compound (TLC Rf = 0.35 in the same solvent mixture) with a yield of 66 mg, 25%. 1H-NMR (Fig. S4,† 400 MHz, CDCl3): δ 8.73 (m, 1H, H′3), 8.45 (m, 1H, H′4), 7.42 (m, 1H, H′5), 5.56 (d, 1H, H1), 5.49–5.24 (m), 4.10–3.42 (m), 1.97–2.16 (m, 54H, COCH3). 13C-NMR (Fig. S5,† 100.6 MHz, CDCl3): δ 170.76, 170.60, 170.41, 170.36, 170.34, 170.24, 170.17, 170.12, 170.09, 170.01, 169.97, 169.88, 169.85, 169.71, 169.63, 169.45, 168.97, 168.68, 168.64 (18 × CO-Ac), 153.32 (C′3), 142.03 (C′4), 140.08 (C′5), 128.63 (C′1), 121.43 (C′2), 116.49 (C′6), 145.58 (C1), 101.07, 100.72, 100.57, 98.24, 97.55, 97.17, 95.94 (C2, 6 × C1), 75.66, 75.36, 74.85, 74.78, 74.45, 74.09, 73.42, 7.38, 72.94, 72.29, 72.06, 71.90, 71.80, 71.70, 70.92, 70.77, 70.49, 70.44, 69.54, 69.49, 69.36, 69.33, 69.23, 69.18, 69.10, 67.45, 67.17 (6 × C2, 7 × C3, 7 × C4, 7 × C5), 65.87, 61.77, 59.22, 58.85 (4 × C6), 20.58–21.01 (18 × CH3). 19F-NMR (Fig. S6,† 376.5 MHz, CDCl3) −194.75 (ddd, JH2–F2 = 47.7 Hz, JH3–F2 = 15.7 Hz, JH1–F2 = 2.5 Hz, F2). Monoisotopic m/z calculated for C81H103FN2O54Na+: 2009.5257; MALDI-ToF MS found: 2009.4; ESI-HRMS found: 2009.5463.
The 2′4′-dinitrophenyl β-glycoside of 2-deoxy-2-fluoro-XXXG (XXXG(2F)-β-DNP (5)) was produced by Zemplen deprotection of 4 (66 mg) in 10 ml methanol/CH2Cl2 9:1, to which 0.5 equivalents of NaOMe (25% in MeOH) were added. The reaction was stirred at 4 °C and monitored by TLC overnight. The product was purified by flash chromatography using water/isopropanol/ethyl acetate (1:3:4) as the mobile phase. The purified product was re-dissolved in water and freeze-dried to give a pale, fluffy powder in 85% yield (34 mg). 1H-NMR (Fig. S7,† 400 MHz, CDCl3): δ 8.73 (m, 1H, H′3), 8.45 (m, 1H, H′4), 7.42 (m, 1H, H′5), 5.56 (d, 1H, H1), 5.49–5.24 (m), 4.10–3.42 (m), 1.97–2.16 (m, 54H, COCH3). 13C-NMR (Fig. S8,† 100.6 MHz, CDCl3): δ 170.76, 170.60, 170.41, 170.36, 170.34, 170.24, 170.17, 170.12, 170.09, 170.01, 169.97, 169.88, 169.85, 169.71, 169.63, 169.45, 168.97, 168.68, 168.64 (18 × CO-Ac), 153.32 (C′3), 142.03(C′4), 140.08 (C′5), 128.63 (C′1), 121.43 (C′2), 116.49(C′6), 145.58 (C1), 101.07, 100.72, 100.57, 98.24, 97.55, 97.17, 95.94 (C2, 6 × C1), 75.66, 75.36, 74.85, 74.78, 74.45, 74.09, 73.42, 7.38, 72.94, 72.29, 72.06, 71.90, 71.80, 71.70, 70.92, 70.77, 70.49, 70.44, 69.54, 69.49, 69.36, 69.33, 69.23, 69.18, 69.10, 67.45, 67.17 (6 × C2, 7 × C3, 7 × C4, 7 × C5), 65.87, 61.77, 59.22, 58.85 (4 × C6), 20.58–21.01 (18 × CH3). 19F-NMR (Fig. S9,† 376.5 MHz, CDCl3) −201.29 (ddd, JH2–F2 = 51.4 Hz, JH3–F2 = 15.2 Hz, JH1–F2 = 2.5 Hz, F2). Monoisotopic m/z calculated for C45H67FN2O36Na+: 1253.3360; MALDI-ToF MS found: 1253.3; ESI-HRMS found: 1253.3354.
The catalytic acid/base mutant CjGH5D(E255A) was generated using the PCR-based QuickChange II Site-Directed Mutagenesis Kit (Agilent, USA) following the manufacturer's protocol. PCR amplification was conducted using the forward primer 5′-TTTGCCGGCACTAACGCCCCCAATGCGGAAAAT-3′ and the reverse primer 5′-ATTTTCCGCATTGGGGGCGTTAGTGCCGGCAAA-3′, utilizing pET28a::CjGH5D46 as the template. The resulting plasmid pET28a:: CjGH5D(E255A) was sequenced to confirm the desired mutation.
Chemically competent E. coli Rosetta DE3 cells were transformed with the plasmid pET28a:: CjGH5D(E255A). The resulting colonies were grown on LB solid medium containing kanamycin (50 μg mL−1) and chloramphenicol (30 μg mL−1). Gene overexpression and recombinant protein purification were then performed as described for the wild-type enzyme.46 The concentration of the purified recombinant CjGH5D(E255A) was determined using an Epoch Micro-Volume Spectrophotometer System (BioTek®, USA) at 280 nm. The presence of the desired mutation was confirmed by intact protein mass spectrometry.49 The purified protein was aliquoted and stored at −80 °C until needed.
To measure time-dependent enzyme inactivation, 2.5 mM of XXXG(2F)-β-DNP (5) was incubated with 20 μM CjGH5D in 50 mM sodium phosphate buffer, pH 7.5, in a total volume of 100 μL (also including 0.1 mg mL−1 bovine serum albumin (BSA) to prevent non-specific loss of activity) for a period of 420 minutes at 40 °C. A control experiment was run in parallel, in which inhibitor was omitted from the buffered enzyme/BSA solution. Periodically, 10 μL of each solution was withdrawn and diluted 1:100 in 50 mM sodium phosphate buffer, pH 7.5, and 100 μL of the diluted solution was added to 100 μL of 0.4 mM XXXG-β-CNP,41 which was previously dissolved in water and preincubated at 40 °C. Linear initial-rate kinetics of 2-chloro-4-nitrophenolate release were measured at 405 nm (ε = 17.74 mM−1 cm−1) over 1 min in a quartz cuvette (l = 1 cm) maintained at 40 °C, essentially as previously described.46
Prior to measuring burst kinetics, the background hydrolysis rate of 235 μL of 5 mM XXXG(2F)-β DNP (5) in 50 mM phosphate buffer, pH 7.5, was measured at 40 °C for 75 min (ε405 = 11.17 mM−1 cm−1, determined using a standard curve of absorbance as a function of dinitrophenolate concentration at the specified buffer concentration and pH conditions. To make the standard solution, 2,4-dinitrophenol was desiccated overnight in presence of phosphorus pentoxide, and thereafter dissolved in the specified buffer). Thereafter, CjGH5D (15 μL, 0.45 mM), which had been preincubated at 40 °C in the same buffer, was added to the cuvette and mixed rapidly (final volume 250 μL, final enzyme concentration 27 μM). The rate of dinitrophenolate (DNP−) release was monitored for an additional 700 min and eqn (1)25 was fit to the data using Origin 8 software, according to the inhibition mechanism shown in Scheme 1b. The cuvette was covered with parafilm to limit evaporation during the entire course of the experiment.
[DNP−] = [DNP−]0(1−e−k2t) + k3t | (1) |
Data were collected at Diamond beamline IO3 and processed using XDS50 for the co-crystallised complex and collected at IO4-1 and processed using DIALS51 for the soaked complex. Both datasets were put through the data reduction pipeline in ccp4i252 which uses POINTLESS, AIMLESS and CTRUNCATE,53 and cut-off at resolutions of 1.7 and 2.0 Å (judged by Rpim < 0.60 and CC1/2 > 0.50 in outer bin), respectively (ESI Table S1†). Both structures were solved using the unliganded structure of the wild-type enzyme (PDB ID 5OYC) as the model for refinement with REFMAC.54 After refinement with water molecules added, the ligands were built into difference electron density in the weighted 2Fo − Fc maps using COOT,55 and validated using PRIVATEER56 prior to deposition at the Protein Data Bank with accession codes 6HAA and 6HA9.
The crucial step of installing fluorine at the 2-position of the protected XXXG glycal was achieved using the electrophilic fluorinating agent Selectfluor™, followed by hydrolytic work-up to obtain the corresponding protected 1-hydroxy-2-deoxy-2-fluoro-oligosaccharide. Selectfluor™ has been widely used for the regiospecific 2-fluorination of a number of glycals.57 In the case of D-glucal, an equimolar mixture of gluco and manno 2F-epimers was obtained, while in the case of the corresponding disaccharides, e.g. cellobiose, the amount of the 2F-gluco epimer reached 80% in polar solvents.44,45 In our case, we were pleased to discover that only the desired gluco configured product was obtained in dry nitromethane,43 albeit as a mixture of α/β anomers, as indicated by 19F NMR (trace amounts of manno epimers were perhaps also present,45 Fig. S3†). As this represents the only example, to our knowledge, of the application of Selectfluor™ to such a large oligosaccharide glucal, the origins of this high stereoselectivity are not fully clear, but may be based in increased local steric congestion or altered access to boat-conformer addition products.45
The reaction of the anomeric mixture with 2,4-dinitrofluorobenzene in the presence of DABCO yielded exclusively the desired β-configured, kinetic product 4,58,59 as ascertained by the large H1–H2 coupling of 7.6 Hz and F2–H1 coupling of 3.0 Hz. With the key stereochemistry set, careful Zemplen deprotection followed by column chromatography yielded pure 5.
Further evidence to suggest turnover of the glycosyl enzyme was obtained by the observation of apparent pre-steady-state burst kinetics, by monitoring the release of 2,4-dinitrophenolate over time (Fig. 2b). The biphasic data were fit by eqn (1), which describes an initial pseudo-first-order exponential accumulation of the fluoroglycosyl-enzyme, followed by a linear phase dominated by steady-state turnover of the covalent intermediate (Fig. 2b).25 After accounting for an effectively linear background hydrolysis rate of 5 in buffer (0.28 × 10−3 min−1), values for k2 and k3 of 7.83 × 10−3 min−1 and 0.16 × 10−3 min−1, respectively, were obtained. The concentration of enzyme active sites, [Eo] = 17.60 μM, obtained from the magnitude of the burst (A405 = 0.20, DNP ε405 = 11.17 mM−1 cm−1) suggested that the CjGH5D preparation was not fully active ([P] = 27 μM in the assay).
In the co-crystallized complex, electron density corresponding to the near-complete glycone was observed spanning the −4 to −1 subsites61 within the active-site cleft, with the exception of the non-reducing-terminal xylosyl residue that was not modelled. In keeping with the mechanism-based design of the inhibitor, the electron density clearly revealed the covalent attachment of C1 of the 2-fluoroglucosyl residue in subsite −1 with Oε1 of the catalytic nucleophile Glu390 (distance ∼1.4 Å). The 2-fluoroglucosyl residue was in a relaxed 4C1 conformation, while the sidechain of Glu390 was rotated from its position in the apo enzyme structure, including an average 1.1 Å translation of Oε1 toward the sugar ring (Fig. 4).
Fig. 4 Active-site superposition of CjGH5D and corresponding covalent inhibitor complexes. a. Full view b. Close-up of the −1 subsite. Side chains are shown with carbon atoms coloured as follows: XXXG-NHAc-CjGH5D covalent complex (PDB ID: 5OYD) in coral, and XXXG(2F)-CjGH5D(E255A) covalent complex (PDB ID: 6HAA) in ice blue, and with catalytic residue carbon atoms shown in pale crimson and light blue respectively. Main chain atoms are shown for E255A for clarity. |
We previously determined the structure of a covalent complex of wild-type CjGH5D with the N-bromoacetylglycosylamine inhibitor XXXG-NHAcBr, in which the catalytic acid–base residue was labelled by substitution of the bromide (PDB ID 5OYD).46 Superposition of XXXG-NHAc-CjGH5D with the XXXG(2F)-CjGH5D(E255A) glycosyl-enzyme complex revealed the basis for the distinct specificity of the two inhibitor types (Fig. 4). Whereas the anomeric carbon of Glc-1 in the XXXG(2F)-β-DNP mechanism-based inhibitor was situated directly above the catalytic nucleophile (Glu390), the three-atom extension of the N-bromoacetyl group placed the α-carbon of the “warhead” in a position suitable for side-on attack by the catalytic acid/base (Glu255), and effectively out of reach of Glu390. Moreover, this latter reaction required the displacement of the sidechain of Glu255 (Cδ) by an additional 2 Å away from the ring atoms of Glc-1 to accommodate the reactive moiety. These steric and sidechain plasticity requirements are likely to underlie the current and previous observations that N-bromoacetylglycosylamine reagents tend to label catalytic acid/base residues in glycosidases (except in the case of off-active-site labelling62).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ob02250j |
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