Tài liệu Báo cáo Y học: The glucose-specific carrier of the Escherichia coli phosphotransferase system Synthesis of selective inhibitors and inactivation studies pptx

Eur J Biochem 269, 4969–4980 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03197.x The glucose-specific carrier of the Escherichia coli phosphotransferase system Synthesis of selective inhibitors and inactivation studies ´ Luis Fernando Garcıa-Alles, Vera Navdaeva, Simon Haenni and Bernhard Erni Departement fuăr Chemie und Biochemie, Universitaăt Bern, Freiestrasse 3, CH-3012, Bern, Switzerland Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate–sugar phosphotransferase system (PTS) 2¢,3¢-Epoxypropyl b-D-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter 3a is the only analogue that inhibits EIIGlc by binding to the highaffinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter The most potent inhibitor in vitro, methyl 6,7-anhydro-D,L-glycero-a-Dgluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl a-D-glucopyranoside (1a) and methyl a-D-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-a-Dglucopyranoside (1e), b-D-glucopyranosyl isothiocyanate (3c) and b-D-glucopyranosyl phenyl isothiocyanate (3d) Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421 Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter Escherichia coli has two transporters for glucose, EIIGlc (IIAGlc-IICBGlc) [1] and EIIMan (IIABMan-IICMan-IIDMan) [2,3], which mediate uptake concomitant with phosphorylation of their substrates The immediate source of highenergy phosphate is the phosphoryl carrier protein HPr which in turn is phosphorylated by phosphoenolpyruvate in a reaction catalysed by enzyme I (EI) EI and HPr together with the carbohydrate transporters (enzymes II, EIIsugar) of diverse specificity and structure are components of the bacterial phosphoenolpyruvate–sugar phosphotransferase system (PTS) [4] The PTS in addition comprises a number of proteins that act as allosteric regulators of enzymes and/ or transcription factors The PTS transporters are homodimers, as indicated by cross-linking, ultracentrifugation, gel filtration, interallelic complementation and cryo-electron crystallography [5–9] One protomer comprises three (or four) functional units, IIA, IIB and IIC(IID), which occur either as protein subunits or as domains in polypeptide chains IIA and IIB sequentially transfer phosphoryl groups from HPr to the transported sugars IIC contains the major determinants for sugar recognition and translocation, as inferred from binding studies [10] and the substrate selectivity of a chimeric EIIGlcNAc/Glc [11] EI, HPr and IIA are phosphorylated at His, whereas IIB domains are phosphorylated at Cys421 in EIIGlc and at His175 in EIIMan EIIGlc is specific for Glc, but EIIMan has a broader substrate specificity for Glc, Man, and other derivatives of Glc altered at the C-2 carbon Both transporters phosphorylate their hexose substrates at OH-6 In spite of their overlapping substrate specificity and analogous mechanism of action, EIIGlc and EIIMan not share amino-acid sequence similarity, and, as judged by the known X-ray structures of their cytoplasmic domains, also assume completely different folds (for a review see [12]) The topology of the membrane-spanning units IICGlc and IICMan-IIDMan are also different, as judged by the characterization of protein fusions between C-terminally truncated IIC(D) domains with alkaline phosphatase and b-galactosidase [13,14] Whereas the sites of EII phosphorylation are known and easily recognized from the invariant amino-acid sequence motifs, residues participating in sugar binding have not been identified Each protomer has been proposed to have a sugar-binding site of its own with the two sites in the dimer being distinguished by their different affinity for the substrate [15] Both sites are simultaneously accessible from the cytoplasmic face The IICBGlc subunits co-operate in so Correspondence to L F Garcı´ a Alles, Departement fur Chemie und ă Biochemie, Universitat Bern, Freiestrasse 3, CH-3012 Bern, ă Schweiz Fax: + 41 31 631 48 87, Tel.: + 41 31 631 37 92, E-mail: garcia@ibc.unibe.ch Abbreviations: PTS, phosphoenolpyruvate–sugar phosphotransferase system; aMGlc, methyl a-D-glucopyranoside; 2dGlc, 2-deoxy-D-glucose; IC50, half inhibitory concentration; FC, flash chromatography (Received June 2002, revised 16 August 2002, accepted 21 August 2002) Keywords: binding site; carbohydrate chemistry; cysteine; glucose transporter; irreversible inhibitor Ó FEBS 2002 4970 L F Garcı´ a-Alles et al (Eur J Biochem 269) Scheme far as phosphoryl transfer from Cys421 on the IIB domain of one subunit to Glc bound to the IIC domain of the second subunit is possible [7] However, whether and how the substrate-binding sites on the IIC domains reorient themselves with respect to the extracellular and cytoplasmic compartment, and how they interact with phosphorylation domains (IIB), is the objective of continuing research With the aim of finding selective irreversible inhibitors of the Glc-specific transporters EIIGlc and EIIMan and of eventually identifying their substrate-binding sites, 13 glucose analogues have been synthesized (compounds 1a)3d, Scheme 1) Epoxides, a-halocarbonyls, isothiocynates or a,b-unsaturated esters were introduced at C-1, where modifications are expected to be tolerated by EIIGlc [15], and at C-6 because of its presumed proximity to the catalytic residues in the active site of the transporter Similarly modified carbohydrates have been used previously to study sugar-recognizing enzymes, e.g epoxypropyl derivatives of N-acetyl-D-glucosamine to label lysozyme [16], maltosyl isothiocyanate to label the human erythrocyte Glc transporter [17], and N-bromoacetylglucosamine to label hexokinase [18] The glucose analogues have been characterized as pseudosubstrates and as reversible and irreversible inhibitors of EIIGlc and EIIMan Two assays were employed: (a) nonvectorial phosphorylation of Glc (analogues) by solubilized and purified EII and by EII-containing membrane fractions – this in vitro assay was used to examine how sugar recognition is effected from the cytoplasmic side of the transporters (a consequence of the inside-out orientation of EII in membrane vesicles); (b) inhibition of Glc uptake by starved cells expressing either EIIGlc or EIIMan These experiments served to study binding to EII from the periplasmic space MATERIALS AND METHODS Materials, bacterial strains and proteins Starting materials for the preparation of compounds 1a)3d, and other components were purchased from commercial sources as specified previously [15] Organic solvents of the highest purity available were dried following standard procedures The membrane transporters were overexpressed and purified from an Escherichia coli K12 strain ZSC112LDG (glk manZ DptsG:Cm) [19] The plasmid pTSGH11 encodes under the control of Ptac a IICBGlc with a C-terminal hexahistidine tag [7] The plasmid pJFLPM encodes the three subunits of the Escherichia coli mannose transporter under the control of the Ptac promoter [20] Membranes containing EIIGlc and EIIMan, and purified EIIGlc, EI, HPr, IIAGlc and IIABMan were prepared as described in [15] In vitro phosphotransferase assays Pyruvate evolution was measured spectrophotometrically in 96-well microtiter plates at 30 °C, using the coupled assay with L-lactate dehydrogenase and NADH Final assay concentrations were: 0.5 lM EI, lM HPr, 15 lM IIAGlc (or 0.5 lM IIABMan) and 0.0013 lLỈlL)1 membrane extract Other conditions were as described in [15] Typical background activities of lmolỈmin)1 were measured in the absence of sugar They were subtracted before subsequent calculations Half inhibitory concentrations (IC50) were determined by measuring the phosphorylation rate of 0.5 mM D-Glc, in the presence of 0–5 mM concentrations of the inhibitors Glc6P was detected in these experiments using Glc6P dehydrogenase (1 mL)1) and mM NADP [21] Fitting of kinetic data using DYNAFIT This software is available free of charge at http://www biokin.com [22] The kinetic constants were estimated as reported [15] Inactivation of nonvectorial phosphorylation A 10-lL sample of inhibitor in buffer A [50 mM Hepes, pH 7.5, mM dithiothreitol, mM MgCl2, mgỈmL)1 BSA, 0.5 mgỈmL)1 egg yolk lecithin (Sigma)] was preincubated for 10 at 30 °C, and then added to 40–60 lL purified IICBGlc in buffer A (final concentration: 10–3 lM) Then 10-lL aliquots were withdrawn at intervals and diluted into 290 lL buffer A at °C The diluted IICBGlc samples were assayed for in vitro PTS activity using the D-Glc6P dehydrogenase assay The final concentrations in the activity assay were 0.08–0.04 lM IICBGlc and mM Glc In vivo transport inhibition Uptake of [14C]methyl a-D-glucopyranoside ([14C]aMGlc) by starved E coli K12 ZSC112LDG cells expressing EIIGlc or of 2-deoxy-D-[14C]glucose ([14C]2dGlc) by starved cells Ó FEBS 2002 expressing EIIMan was assayed as described previously [15] Transport rates were calculated from the amount of [14C]sugar accumulated inside the cells, typically after 5, 15, 25, 40 and 120 s Inactivation of uptake by starved cells E coli K12 ZSC112LDG cells expressing either EIIGlc or EIIMan were prepared as described previously [15] To 0.65 mL cell suspension in M9 medium (0.2–0.1 gỈmL)1) at room temperature were added 26 lL of a stock solution of the irreversible inhibitor (0.5–0.025 M), with or without Glc (0.25 M) Aliquots (0.15 mL) were withdrawn at the indicated time points and diluted into ice-cold M9 medium (0.85 mL) The cells were collected by centrifugation and resuspended in mL fresh M9 medium The washed cells were then assayed for uptake activity as described above Synthesis of compounds 1a-3d 6-O-Bromoacetyl derivatives (1a,c) [23], methyl 6-deoxy-6isothiocyanato-a-D-glucopyranoside (1e) [24], and b-Dglucopyranosyl isothiocyanate (3c) [25] were prepared by the reported procedures Methyl 6-O-chloroacetyl-a-Dglucopyranoside (1b) was prepared like 1a using chloroacetyl chloride instead of bromoacetyl bromide The yield was 56% after flash chromatography (FC) (ethyl acetate/methanol, 96 : 4, v/v): 1H NMR (CD3OD) d: 4.66 (1H, d, J ¼ 3.7 Hz, H1), 4.47 (1H, dd, J ¼ 11.8, 2.2 Hz), 4.31 (1H, dd, J ¼ 11.8, 6.3 Hz), 4.24 (2H, s, CH2Cl), 3.73 (1H, m), 3.61 (1H, dd, J ¼ 9.5, 8.8 Hz), 3.40 (3H, s CH3O), 3.39 (1H, overlapped), 3.30 (1H, m) 13C NMR (CD3OD) d: 169.1, 101.3, 75.1, 73.5, 71.8, 70.9, 66.3, 55.7, 41.7 MS (ESI) m/z 293 ([M + Na]+, 40%) Methyl 2,3-anhydro-a-D-allopyranoside (2a) and methyl 3,4-anhydro-a-D-galactopyranoside (2b) were obtained by desilylation of mmol of the 6-O-[dimethyl-(1,1,2-trimethylpropyl)silyl]-protected forms [26] with CsF (3 mmol) in dimethylformamide (50 mL) at 110 °C, for 30 Evaporation in vacuo of dimethylformamide, and FC (ethyl acetate/methanol, 96 : 4, v/v) afforded 2a in 81% yield [26] and 2b in 48% yield [27] b-D-Glucopyranosyl phenyl isothiocyanate (3d) was purchased (Sigma) Preparation of methyl 6,7-anhydro-D,L-glycero-a-Dglucoheptopyranoside (1d) Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside (4) was obtained from methyl a-D-glucopyranoside, following conventional sugar transformations [28] A solution of (2.6 g, 5.6 mmol) in 12 mL dichloromethane was added dropwise ˚ to a suspension of activated powdered A molecular sieves (11 g) and pyridinium chlorochromate (5.6 g, 26 mmol) in dry dichloromethane (80 mL) The resulting mixture was stirred for 10 at room temperature, 80 mL hexane was added, and the mixture was filtered through a pad of silica gel Elution with ethyl acetate/hexane (1 : 1, v/v) and concentration furnished 1.92 g (75%) methyl 2,3,4-triO-benzyl-a-D-glucohexodialdo-1,5-pyranoside (5) [29] The aldehyde (1 g, 2.1 mmol, in mL of dry tetrahydrofurane) was slowly added under argon to a flask containing methyltriphenylphosphonium ylide (3.4 mmol) in 40 mL dry tetrahydrofurane at )78 °C [30] After 15 min, the Inhibition of EIIGlc (Eur J Biochem 269) 4971 cooling bath was removed and stirring was continued for 1.5 h The reaction was stopped at °C by the addition of 10 mL methanol After concentration, 150 mL diethyl ether was added The solution was washed with brine (2 · 50 mL), and the organic phase dried over MgSO4 Concentration followed by FC (hexane/ethyl acetate : 2, v/v) gave 0.69 g (70%) methyl 6,7-dideoxy-2,3,4-tri-O-benzyl-a-D-glucohept-6-enopyranoside (6) [31] A solution of compound (0.2 g, 0.43 mmol) in mL dry dichloromethane was stirred at room temperature with 3-chloroperoxybenzoic acid (0.5 g, 2.9 mmol) for 15 h Diethyl ether (50 mL) was added and the solution was washed with 0.12 M aqueous Na2S2O3 (3 · 20 mL), saturated NaHCO3 (2 · 20 mL) and brine (20 mL) The ether phase was dried over MgSO4, filtered and evaporated, yielding 0.18 g of the mixture of epoxides (7), 92% Compound (0.12 g, 0.25 mmol) in mL 4.4% formic acid in methanol was added to a suspension of 0.3 g palladium black in mL 4.4% formic acid in methanol [32] After 30 min, the catalyst was removed by filtration through celite, and washing with methanol (2 · mL) After evaporation of the solvent, 76 mg (81%) of the diastereomeric mixture of 1d was recovered 1H NMR (CD3OD) d: 4.85 (1H, m, H1), 3.92–3.64 (3H,m), 3.60–3.30 (3H, m), 3.55 (3H, s CH3O), 3.05–2.87 (1H, m) 13C NMR (CD3OD) d: 101.7, 75.5, 75.4, 74.2, 73.8, 73.6, 73.5, 56.1, 53.7, 53.5, 45.8, 44.8 MS (ESI) m/z 229 ([M + Na]+, 100%) Synthesis of methyl (6E )-6,7-dideoxy-a-D-gluco-oct-6enopyranosiduronic acid (1g) and its methyl ester (1f) Methyl 2,3,4-tris-O-(trimethylsilyl)-a-D-gluco-hexodialdo1,5-pyranoside (9) (0.3 g, 0.73 mmol), prepared as described in ref [15], was olefinated at room temperature with methyl triphenylphosphoranylidene acetate (0.33 g, mmol) in mL dry dichloroethane After h reaction, the solvent was removed by evaporation, and 10 was purified by FC (hexane/ethyl acetate, 92 : 8, v/v): 0.2 g, 44% yield Compound 10 (0.1 g, 0.21 mmol) was desilylated by stirring for h at room temperature with methanol (2 mL) and K2CO3 (2 mg) After evaporation 54 mg 1f (100%) was obtained : H NMR (CD3OD) d: 7.29 (1H, dd, J ¼ 15.8, 4.4 Hz, H6), 6.31 (1H, dd, J ¼ 15.8, 1.8 Hz, H7), 4.93 (1H, d, J ¼ 4.0 Hz, H1), 4.13 (1H, ddd, J ¼ 9.9, 4.4, 1.5 Hz, H5), 3.84 (1H, dd, J ¼ 9.6, 8.8 Hz, H3), 3.61 (1H, dd, J ¼ 9.6, 3.7 Hz, H2), 3.58 (3H, s, CH3O), 3.52 (3H, s, CH3O), 3.29 (1H, ddd, J ¼ 9.9, 8.8, 1.1 Hz, H4); 13C NMR (CD3OD) d: 168.8, 147.0, 122.1, 101.8, 75.7, 75.4, 73.7, 71.8, 56.2, 52.4 MS (ESI) m/z 287 ([M + K]+, 100%) Hydrolysis of 1f (23 mg, 0.09 mmol, 0.1 M solution in water) was effected at pH 12 (1 M KOH) for h The derivative 1g was obtained as a potassium salt in quantitative yield: 1H NMR (D2O, pD 7) d: 6.51 (1H, dd, J ¼ 15.8, 7.0 Hz, H6), 6.11 (1H, dd, J ¼ 15.8, 1.1 Hz, H7), 4.80 (1H, d, J ¼ 3.7 Hz, H1, overlapped with water signal), 4.14 (1H, dd, J ¼ 9.6, 7.0 Hz, H5), 3.70–3.58 (2H, m), 3.41 (3H, s, CH3O), 3.33 (1H, dd, J ¼ 9.6, 8.8, 1.1 Hz, H4); 13C NMR (D2O, pD 7) d: 177.2, 140.6, 133.5, 102.1, 75.2, 75.4, 73.8, 73.7, 57.9 MS (ESI) m/z 233 ([M-H]–, 100%) Synthesis of 2¢,3¢-epoxypropyl b-D-glucopyranoside (3a) The allyl b-D-glucopyranoside (12) was prepared by reaction of acetobromoglucose (11) with allyl alcohol [33] An ice-cooled solution of 12 (90 mg, 0.23 mmol) in Ó FEBS 2002 4972 L F Garcı´ a-Alles et al (Eur J Biochem 269) mL dichloroethane was treated with freshly prepared dimethyldioxirane in acetone (3 mL,  0.3 mmol) [34] After h, the ice bath was removed, and dimethyldioxirane (2 mL) was added after and h The reaction was continued overnight Solvent was removed by evaporation, and the resulting epoxypropyl (13) (93 mg) was deacetylated as described [35], to furnish 3a as a : diastereomeric mixture: 1H NMR (CD3OD) d: 4.52 (0.4H, d, J ¼ 7.7 Hz, H1), 4.49 (0.6H, d, J ¼ 7.7 Hz, H1), 4.31– 3.68 (4H, m), 3.55–3.38 (5H, m), 2.98 (1H, m, CH2 epoxide), 2.88–2.81 (1H, m, CH2 epoxide); 13C NMR (CD3OD) d: 104.4, 104.3, 77.9, 75.1, 75.0, 71.6, 71.5, 71.4, 71.2, 62.8, 62.7, 52.0, 51.8, 45.2, 45.0 MS (ESI) m/z 259 ([M + Na]+, 100%) [35] The diastereomeric mixture 3a was also prepared by following an alternative route Tri-O-acetyl-D-glucal (15) (3 g, 10.8 mmol) was refluxed with benzyl chloride (24 mL), KOH (9.4 g) and toluene (20 mL) After h, the solution was concentrated in vacuo Then 150 mL diethyl ether was added and the solution was washed with water (2 · 100 mL) and saturated NaHCO3 (100 mL) The organic phase was dried over MgSO4, filtered, and concentrated The residue was chromatographed (hexane/diethyl ether, : 3, v/v) giving 2.3 g (52%) of 16 The epoxide 17 was then prepared from 16 by reaction with dimethyldioxirane, as described [36] The derivative 17 (0.24 g, 0.55 mmol) was treated with mL racemic glycidol at room temperature After h reaction, the excess glycidol was removed under vacuum, and the residue was chromatographed with diethyl ether, giving 0.13 g of 14 [37] The epoxide 14 (50 mg, 0.1 mmol) was debenzylated in 30 by following the same method as for 1d 23 mg of a : diastereomeric mixture of 3a was obtained Preparation of chloroacetyl b-D-glucopyranoside (3b) The epoxide 17 (0.2 g, 0.46 mmol) was treated with a solution of chloroacetic acid (0.11 g, 1.15 mmol) in dry dichloromethane (10 mL) The mixture was stirred at room temperature overnight Evaporation and FC (hexane/ diethyl ether, : 1, v/v) furnished 0.17 g (70%) chloroacetyl 3,4,6-tri-O-benzyl-b-D-glucopyranoside (18) Removal of the benzyl groups, as described for compound 1d (see above), and FC (ethyl acetate/methanol, : 1, v/v) resulted in 73 mg (88%) compound 3b: 1H NMR (CD3OD) d: 5.73 (1H, d, J ¼ 7.7 Hz, H1), 4.48 (2H, s, CH2Cl), 4.03 (1H, dd, J ¼ 12.1, 2.8 Hz, H6a), 3.86 (1H, dd, J ¼ 12.1, 4.8 Hz, H6b), 3.63–3.46 (4H, m); 13C NMR (CD3OD) d: 168.1, 96.8, 78.9, 77.8, 73.9, 70.9, 62.2, 41.6; MS (ESI) m/z 279 ([M + Na]+, 100%) RESULTS Synthesis of inhibitors The synthesis and characterization of compounds 1b, 1d, 1f, 1g and 3b (Scheme 1) is reported for the first time, and the epoxypropyl derivative 3a is prepared by a new route All other compounds of Scheme were synthesized following described procedures, with modifications as specified in Materials and Methods All compounds were characterized by 1H-NMR and 13C-NMR spectroscopy and by electrospray MS Scheme The epoxide 1d was prepared in seven steps from methyl a-D-glucopyranoside (aMGlc, Scheme 2) Conventional procedures were followed for the synthesis of the C-6 hydroxyl-free analogue [28]: (a) selective protection of the 6-hydroxy group by reaction with trityl chloride, (b) benzylation of the 2, 3, 4-OH groups, and (c) acid-catalysed removal of the 6-O-trityl group Oxidation of the free C-6 hydroxymethylene of to aldehyde with pyridinium chlorochromate, followed by Wittig methylenation at C-6, epoxidation of the newly created double bond of with 3-chloroperoxybenzoic acid, and removal of the protecting benzyl groups present in by catalytic transfer hydrogenation led to the epoxide 1d This compound was obtained as a C-6 diastereomeric mixture which was used without further separation The a,b-unsaturated methyl ester 1f and its free carboxylic acid 1g were synthesized as depicted in Scheme 3, as described previously [38] and the modifications which were recently introduced for the preparation of C-6 aldehyde derivatives of Glc [15] The key step is the use of Collins reagent for the selective oxidation of the primary trimethylsilyl ether of the fully silylated monosaccharide to an aldehyde (step ii) The resulting 2,3,4-tris-trimethylsilylated derivative was then condensed with methyl triphenylphosphoranylidene acetate to the a,b-unsaturated methyl ester 10 This reaction produced exclusively the E-isomer, as judged from the value of the NMR coupling constant between the protons connected to the double bond (3JH6-H7 ¼ 15.8 Hz) Removal of the trimethylsilyl groups afforded the methyl ester 1f This compound was hydrolyzed under controlled alkaline conditions to the potassium salt of the carboxylic acid 1g The epoxypropyl and chloroacetyl derivatives 3a and 3b were prepared as shown in Scheme The epoxypropyl derivative was synthesized via two routes (a) The allyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside 12 was first obtained by silver oxide promoted nucleophilic substitution Ó FEBS 2002 Inhibition of EIIGlc (Eur J Biochem 269) 4973 at C-1 of acetobromoglucose (11) and then reacted with dimethyldioxirane to afford the epoxide 13 This compound was deacetylated in the presence of catalytic amounts of methanolic sodium methoxide, giving the mixture of C-1 diastereomers 3a (b) Tri-O-benzyl-D-glucal (16) was converted into the reactive a-epoxide 17 by reaction with dimethyldioxirane [36] The oxirane ring was then opened by treatment with (±)-glycidol to afford the pure b anomer 14 In a similar manner, the pure chloroacetyl b-D-glucopyranoside 18 was obtained by reaction between 17 and chloroacetic acid Removal of the protecting benzyl groups of 14 and 18 by catalytic transfer hydrogenation resulted in compounds 3a and 3b, respectively Glucose analogues as pseudosubstrates of EIIGlc and EIIMan Scheme Compounds 1a)3d were assayed in vitro as substrates of the two PTS transporters Phosphoenolpyruvate-dependent phosphotransferase activity was monitored by coupling the formation of pyruvate (evolved from phosphoenolpyruvate) with its reduction to lactate catalysed by L-lactate dehydrogenase The C-1 epoxypropyl derivative 3a was the only one out of the 13 glucose analogues that functioned as a good substrate of EIIGlc The apparent Km of EIIGlc for 3a is 28 lM, which is comparable to the 60 lM value determined in a parallel experiment for Glc (Table 1) Vmax of EIIGlc for 3a was 14.9 lMỈmin)1 which is only three times slower than for Glc (40 lMỈmin)1) Compound 3a is highly selective for EIIGlc and is not phosphorylated by EIIMan The C-1 chloroacetyl derivative 3b induced a slow consumption of NADH in the presence of L-lactate dehydrogenase, but also formation of NADPH in the presence of Glc6P dehydrogenase This background activity must therefore be due to phosphorylation of Glc released by slow hydrolysis of 3b, and not to phosphorylation of intact 3b The C-1 isothiocyanate (3c) coexists in a : ratio with the 1,2-cyclic thiocarbamate form [25], neither of which was a substrate of EIIGlc or EIIMan The bulky C-1 phenyl isothiocyanate 3d and the epoxides 2a and 2b were not substrates This confirms the earlier observation that OH-2, OH-3 and OH-4 are essential for recognition and that a distortion of the pyranose ring by the epoxide ring is not tolerated [15] Compounds 1a–g are modified at C-6 and therefore cannot be phosphorylated Glucose analogues as reversible inhibitors of EIIGlc and EIIMan Scheme Compounds 1a)3d were assayed in vitro as inhibitors of Glc phosphorylation by the two PTS transporters The concentration of the glucose analogues was varied between and mM while the substrate, D-Glc, was kept constant at 0.5 mM To minimize the effect of potential time-dependent irreversible inactivation, the assays were started by the simultaneous addition of Glc and the inhibitor Phosphorylation of Glc was measured with the Glc6P dehydrogenasecoupled assay Representative data for three compounds are shown in Fig 1, and IC50 of all compounds are listed in Table Without exception, EIIGlc was more strongly inhibited than EIIMan The C-6 epoxide 1d was the strongest inhibitor It inhibited EIIGlc with an IC50 of 0.07 mM, but had almost no effect on EIIMan This result was confirmed Ó FEBS 2002 4974 L F Garcı´ a-Alles et al (Eur J Biochem 269) Table Kinetic constants of EIIGlc and EIIMan for analogues of D-glucose Phosphorylation was measured at 30 °C with the L-lactate dehydrogenase-coupled assay Kinetic constants were derived from a best fit to a Michaelis–Menten hyperbola NS, No saturation observed EIIGlc EIIMan Substrate Vmaxa (lMỈmin)1) Km (lM) Vmax/Kma ( · 103 min)1) Vmaxa (lMỈmin)1) Km (lM) Vmax/Kma ( · 103 min)1) Glc 3a 3bb 40 ± 14.9 ± 0.6 35 ± 60 ± 10 28 ± 700 ± 100 700 ± 130 530 ± 110 50 ± 20 32 ± NS 40 ± 10 50 ± 10 NS 1100 ± 400 680 ± 200 7±2 37 ± 20 Using 0.0013 lLỈlL)1 membrane extract Concentrations of other PTS components are indicated in Materials and methods freshly purified compound The reaction is also detected with the D-Glc6P dehydrogenase assay a with [14C]aMGlc as substrate and direct detection of [14C]aMGlc6P (results not shown) The second best C-6modified analogues, bromoacetyl-Glc (1a), bromoacetylMan (1c), and isothiocyano-Glc (1e) had a 10 times higher IC50 than 1d The epimeric bromoacetyl derivatives 1a and 1c both inhibited EIIGlc, although EIIGlc strongly discriminates between Glc and Man The chemically less reactive chloroacetyl-Glc (1b) did not inhibit EIIGlc This already suggests that inhibition by 1a and 1c might be nonspecific and due to rapid alkylation of Cys421 (see below) Of the analogues modified at C-1, the epoxide 3a (a pseudosubstrate) and the chloroacetyl 3b had an IC50 of mM The remaining analogues with bulky and rigid substituents had IC50 > 2.4 mM or did not inhibit at all Inhibition of Glc phosphorylation by the C-1 and C-6 epoxides 3a and 1d, the two most potent analogues, was examined in more detail EIIGlc-dependent Glc phosphorylation was measured at four different concentrations of 3a and 1d, and the results were plotted in the Eadie–Hofstee b Using form (Fig 2) In the absence of an inhibitor, EIIGlc displayed biphasic kinetics (Fig 2, solid symbols), consistent with the presence of two binding sites of different affinity [15] Addition of the C-6 epoxide 1d (Fig 2A) did not change the biphasic shape In contrast, addition of the phosphorylatable C-1 epoxide 3a (Fig 2B) resulted in a transition from the biphasic to a monophasic shape of the curve The datapoints in Fig were fitted to the two-activesite model that was recently introduced to explain kinetic data collected with several glucose analogues as pseudosubstrates of EIIGlc and EIIMan [15] A high-affinity low-turnover site (represented by E1) and a low-affinity high-turnover site (E2) were proposed to coexist at the cytoplasmic side of EIIGlc According to this model, the estimated ratio of inhibition constant/substrate dissociation constant for the pair 1d and Glc (I/S) was KI2/KS2 ¼ 0.04 at the low-affinity site, and KI1/KS1 ¼ at the high-affinity site The curved shape of the plot and the KI/KS ratios indicate that the C-6 epoxide 1d, like the C-6 aldehydes of Glc and aMGlc [15], preferentially inhibits the low-affinity site of EIIGlc For the phosphorylatable C-1 epoxide 3a (Fig 2B), Table Compounds 1a)3d as inhibitors of D-glucose phosphorylation Phosphorylation of D-Glc (0.5 mM) was measured using the D-Glc6P dehydrogenase-coupled assay at 30 °C in the presence of 0–5 mM concentrations of the inhibitors ND, No significant inhibition detected Half inhibitory concentrations (IC50) are given in mM Values in parentheses determined measuring inhibition of phosphorylation of [14C]a-MGlc (0.5 mM) IC50 Inhibitor Fig Inhibition of nonvectorial phosphorylation Relative rate of Glc phosphorylation by membranes containing EIIGlc (solid symbols) and EIIMan (open symbols) in the presence of inhibitors 1d (squares), 1e (triangles) and 3a (circles) The IC50 values obtained from these and similar plots are listed in Table [Glc] ¼ 0.5 mM Glc phosphorylation was detected with the D-Glc6P dehydrogenase assay EIIGlc EIIMan 1a 1b 1c 1d 1e 1f 1g 2a 2b 3a 3b 3c 3d 0.8 (2) >5 0.8 (3) 0.07a 0.9 (2)  5a > 5a ND ND 1.0a 1.3a >5 2.4 ND ND ND >5 ND ND ND ND ND ND ND ND ND a Confirmed with the radioactivity-based assay Inhibition of EIIGlc (Eur J Biochem 269) 4975 Ó FEBS 2002 consistent with inhibition by a compound that preferentially binds to the high-affinity site The different affinities of the analogues modified at C-1 and C-6 for the two sites also explain the observed difference of their IC50: low for C-6, high for the C-1 epoxides They were determined at 0.5 mM Glc, at which concentration the high-turnover site (low affinity) is saturated and therefore preponderant in catalysis Glucose analogues as irreversible inhibitors of EIIGlc To assay for irreversible inhibition, membrane fractions containing EIIGlc were preincubated with the different compounds 1a)3d at 30 °C Preliminary experiments showed that the extent of inactivation depended on the concentration of dithiothreitol present during the incubation For instance, inactivation of EIIGlc by iodoacetamide was 50% in the presence of 0.5 mM, and almost complete in the presence of mM dithiothreitol (results not shown) For this reason EIIGlc-containing membranes were always preincubated in the presence of mM dithiothreitol Three conditions were assayed: (a) treatment with the inhibitor alone; (b) in the presence of a 10 mM concentration of a protective substrate, glucose (+ Glc, Table 3); (c) in the presence of phosphoenolpyruvate and the soluble PTS proteins necessary to keep the reactive Cys421 of EIIGlc in the phosphorylated state (+ PEP) Aliquots were withdrawn after different time intervals and assayed for glucose phosphotransferase activity Controls without inhibitor were run in parallel to correct for thermal inactivation, which in all cases was less than 10% of the activity at time zero The corrected data were then fitted to decay curves from which the inactivation rates (kinact) under conditions (a) to (c) were calculated Fig Reversible inhibition of EIIGlc Eadie–Hofstee plots of nonvectorial phosphorylation of D-Glc (0–2 mM) by membrane fractions containing EIIGlc Phosphorylation was assayed in the presence of inhibitors 1d [A, lM (squares), 33.3 lM (triangles), 100 lM (circles) and 300 lM (stars)] and 3a [B, mM (squares), 0.33 mM (triangles), mM (circles) and mM (stars)] The lines represent the best global least-squares fit of the data to a kinetic model of EII with two independent enzymatic activities, E1 (high affinity) and E2 (low affinity) [15] Binding of the inhibitor to both E1 and E2 was allowed The kinetic constants obtained from the best fit are: with 1d (A) KS1 ¼ lM, k1 ¼ 23 min)1, KI1 ¼ 19 lM, KS2 ¼ 190 lM, k2 ¼ 39 min)1, KI2 ¼ lM; with 3a (B) KS1 ¼ lM, k1 ¼ 16 min)1, KI1 ¼ lM, KS2 ¼ 140 lM, k2 ¼ 32 min)1, KI2 ¼ 700 lM KS1 and KS2 are the dissociation constants of E1 and E2 for Glc, KI1 and KI2 the dissociation constants for the inhibitor, and k1 and k2 are the turnover numbers DYNAFIT was used to fit the experimental data to the theoretical model and in the subsequent simulations [22] the corresponding ratios were KI2/KS2 ¼ and KI1/ KS1 ¼ 0.7 These values and the transition of the Eadie– Hofstee plot from the biphasic to a monophasic shape are Table Rates of inactivation of IICBGlc Incubation of purified IICBGlc with the indicated concentration (mM) of the analogues 1a)3d was carried out at 30 °C Rate constants (min)1) were calculated by nonlinear fit to a first-order decay function of the form: y ¼ A exp(–kinact t) + residual kinact Inhibitor Concn a-Haloester analogues 1ac 1b 30 1cc 3b IAcNH2 BrAcOH Epoxides 1d 60 2a 60 2b 60 3a 60 Isothiocyanates 1e 15 3c 15 3d a,b-Unsaturated carboxylic acid derivatives 1f 60 1g 60 a + Glca – 1.51 0.12 0.9 0.023 0.61 0.101 ± ± ± ± ± ± 0.09 0.02 0.1 0.006 0.06 0.004 0.007 ± 0.001 < 0.001 < 0.001 0.0038 ± 0.0004 0.35 ± 0.04 0.54 ± 0.04 0.55 ± 0.09 < 0.001 < 0.001 + PEPb 1.5 ± 0.3 0.15 ± 0.02 – – 0.54 ± 0.09 – < < < – < < – – – – – – – – 0.7 ± 0.1 0.58 ± 0.06 0.67 ± 0.06 < 0.001 0.4 ± 0.1 < 0.001 – – – – 0.001 0.001 0.001 0.001 0.001 Incubation in the simultaneous presence of 10 mM Glc b Incubation in the presence of 1.5 mM phosphoenolpyruvate, 0.5 lM E1, 0.5 lM HPr, lM IIAGlc c Rate constants calculated by nonlinear fit to a second-order decay function: y ¼ A/(1 + kinactt) + residual 4976 L F Garcı´ a-Alles et al (Eur J Biochem 269) Representative examples of the inactivation curves obtained with 1a, iodoacetamide and bromoacetic acid are given in Fig 3, and the results obtained with all compounds are listed in Table Rates of inactivation were fastest with the C-6 bromoacetyl analogues 1a and 1c and with the isothiocyanates, more than 10 times slower with the C-6 and C-l chloroacetyl compounds 1b and 3b, and at least 100 times slower for the epoxides The presence of 10 mM Glc did not protect against inactivation To the contrary, the presence of Glc slightly sensitized EIIGlc for inactivation by the isothiocyanates 1e and 3d On the other hand, the rate of inactivation by bromoacetyl-Glc (1a) was 15 times faster than by bromoacetic acid and 2.5 times faster than by the chemically more reactive iodoacetamide, suggesting some specificity and selectivity of the glucose analogues for EIIGlc Phosphorylation of EIIGlc completely protected against inactivation, indicating that Cys421 is the most, if not the only, reactive residue Protection was incomplete in the presence of the C-1 SCN-Glc (3c) which by its free OH-6, at the high EIIGlc concentrations present during the incubation, can accept a phosphoryl group and thereby deprotect Cys421 Fig Irreversible inhibition of EIIGlc A membrane preparation containing EIIGlc was preincubated with mM inhibitor 1a (circles), iodoacetamide (stars) and bromoacetic acid (tickmarks) in the presence of mM dithiothreitol at 30 °C Aliquots were withdrawn after the indicated incubation time, 30-fold diluted into cold buffer, and residual PTS activity was then measured in a standard phosphotransferase assay In the lowest part of the figure are presented the residuals of the fit to exponential (upper panels), second-order (central panels) and biphasic (lower panels) decay curves for inactivation by 1a (circles) and iodoacetamide (stars) Ó FEBS 2002 Inhibition and inactivation of sugar uptake by starved cells Analogues 1a)3d were assayed as competitive inhibitors of [14C]sugar uptake by intact cells The nonmetabolizable [14C]aMG and [14C]2dGlc were used as substrates, instead of [14C]glucose These glucose analogues are selectively transported via EIIGlc and EIIMan, respectively, and consequently further guarantee that uptake is due to the studied transporter The reactive analogues and [14C]aMG or [14C]2dGlc were added in molar ratio of 10 : and the uptake of [14C]aMGlc via EIIGlc or of [14C]2dGlc via EIIMan was measured (Fig 4) The C-1 epoxide 3a was the only analogue that efficiently blocked EIIGlc-dependent uptake It also slightly reduced the rate of EIIMan-mediated uptake of 2dGlc The C-6 epoxide 1d weakly inhibited uptake by EIIGlc only, whereas the other analogues were inactive with both transporters To test for inactivation of transport, starved cells were preincubated with the C-6 bromoacetyl-Glc 1a and bromoacetyl-Man 1c, the C-6 epoxide 1d, the C-1 epoxide 3a, the C-6 isothiocyanate 1e and the C-1 isothiocyanate 3d, and then the residual uptake activity was determined (see Table 4) The C-6 bromoacetyl compounds 1a and 1c completely blocked uptake by EIIGlc and EIIMan Preincubation with 20 mM C-1 phenylisothiocyanate 3d reduced the uptake rate fivefold and 20-fold, respectively The other analogues were less inhibitory Inactivation by the bromoacetyl-Glc (1a) and bromoacetyl-Man (1c) was examined in more detail Taking into account that Glc appeared to sensitize rather than protect EIIGlc for inactivation in vitro (see above), cells were preincubated for and with and without inhibitor in the absence and presence of 10 mM Glc With short incubation times, cells expressing EIIGlc were inactivated slightly faster by the glucose analogue 1a than by the Fig Inhibition of sugar uptake by starved cells EIIGlc-dependent uptake of [14C]aMGlc (0.1 mM, black bars), and EIIMan-dependent uptake of [14C]2dGlc (0.1 mM, grey bars) in the presence of the indicated inhibitors (1 mM) DPTS, Background uptake by a strain lacking both EIIGlc and EIIMan 100% uptake corresponds to 25 nmolỈmin)1Ỉmg)1 dry weight of cells expressing EIIGlc, and 90 nmolỈmin)1Ỉmg)1 cells expressing EIIMan Inhibition of EIIGlc (Eur J Biochem 269) 4977 Ó FEBS 2002 Table Inactivation of sugar uptake by starved cells by compounds 1a-3d Cells were treated with the indicated concentrations (mM) of inhibitor for 60 at room temperature The rate of accumulation of radioactive sugar in the pretreated cells was then measured Uptake rates are in nmolỈmin)1Ỉmg)1 dry weight of cells EIIGlc was measured using [14C]aMGlc (0.1 mM, 6400 d.p.m.Ỉnmol)1) EIIMan was measured using [14C]2dGlc (0.1 mM, 5600 d.p.m.Ỉnmol)1) Uptake rate Inhibitor Concn EIIGlc – 1a 1c 1d – 1 20 20 20 20 23 ± < 0.02 < 0.02 10 ± 4± 16 ± 6± 17 ± 8± 20 ± 5± 1e 3a 3d EIIMan 10 4 41 ± < 0.02 < 0.02 – 19 ± 14 ± 8± – 17 ± 6.4 ± 2± 17 0.8 mannose epimer 1c (Fig 5A) whereas the opposite was true for EIIMan (Fig 5B) This indicates that EIIGlc and EIIMan are, to some extent, selectively inactivated by their cognate substrate analogues As observed above, the presence of Glc did not protect, but to the contrary sensitized, EIIGlc for inactivation (Fig 5A, grey bars) In the presence of Glc, the rates of EIIGlc inactivation by 1a and 1c increased 18-fold and 27-fold, respectively This effect of Glc is specific for Fig Glucose-sensitized inactivation of [14C]sugar uptake by starved cells Cells expressing EIIGlc (A) or EIIMan (B) were incubated for with and without inhibitors (10 mM) in the absence (black bars) and presence (grey bars) of 10 mM D-Glc Cells were washed to remove excess inhibitor and Glc and assayed for uptake activity as described in Materials and methods and in the legend to Fig EIIGlc and the C-6 bromoacetyl sugars It was not observed with the C-1 isothiocyanate 3d and the C-6 epoxide 1d (results not shown), nor with EIIMan (Fig 5B) Antibacterial activity Initially, PTS-specific toxic sugars can be considered as potential antibiotics For that reason, and in view of the results presented above, the analogues 1a)3d were screened as antibacterial agents towards E coli cells expressing either EIIGlc or EIIMan Cell growth in mineral medium supplemented with glucose was monitored spectrophotometrically (550 nm), in the presence of variable concentrations of the glucose analogues The PTS specificity of these compounds was assessed in two ways: (a) also using the background E coli strain lacking both transporters, and (b) studying growth with glycerol as carbon source, instead of glucose Thus, cell growth was prevented or delayed by 1a and 1c (> 0.04 mM concentration required), 1d (> mM), 1e (> 0.2 mM), 3c and 3d (> 0.8 mM) However, none of the analogues showed PTS-mediated antibacterial activity All kinds of cells, expressing the PTS transporters or not, were inhibited to the same extent (not shown) Moreover, the results were independent of whether glucose or glycerol were added as carbon source DISCUSSION Thirteen glucose analogues with a-haloester, isothiocyanate, epoxide and a,b-unsaturated ester functions at positions C-1 and C-6 were synthesized and characterized as pseudosubstrates, reversible and irreversible inhibitors of EIIGlc and EIIMan The C-1 epoxide analogue 3a was the only efficient pseudosubstrate of EIIGlc in vitro, and the only reversible inhibitor of sugar uptake by starved cells The C-6 isothiocyanate 1e and epoxide 1d and the C-1 epoxide 3a and chloroacetate 3b were selective reversible inhibitors of nonvectorial phosphorylation by EIIGlc The C-6 bromoacetylglucose and bromoacetylmannose derivatives 1a,c irreversibly blocked in vitro phosphorylation and uptake by starved cells The isothiocyanates only blocked in vitro phosphorylation by EIIGlc in membrane preparations, but not uptake The C-6 bromoacetyl derivatives and isothiocyanates presumably reacted with the active-site residue Cys421 This cysteine transfers the phosphoryl group from the IIAGlc subunit to the OH-6 of the substrate in a doubledisplacement reaction [39] It is highly exposed at the edge of a split a/b sheet [40], and from this position rapidly quenches the reactive analogues It was expected that this residue would react, but not that it would be the only reactive one It is noteworthy that (a) the rate of inactivation by bromoacetyl-Glc is 2.5 times faster than by the chemically more reactive but unspecific iodoacetamide, (b) EIIGlc is completely protected against inactivation if Cys421 is phosphorylated or converted into a disulfide before exposure to the alkylating analogues (results not shown), and (c) inactivation of EIIGlc is accelerated in the presence of Glc (see below) Although the dominant reactivity of Cys421 compromised the labelling of other active-site residues, the glucose analogues nevertheless provided new, and confirmed recent, insight into (a) the kinetic properties [15], (b) the selectivity, and (c) the conformational coupling of the EIIGlc active sites Ó FEBS 2002 4978 L F Garcı´ a-Alles et al (Eur J Biochem 269) Fig Proposed model for the IICBGlc dimer of EIIGlc IICBGlc consists of a membrane-spanning C domain (grey) and the cytoplasmic IIB domain (black) IICBGlc is phosphorylated at Cys421 by the soluble IIAGlc subunit It is proposed that two nonvectorial phosphorylation sites are present at the cytoplasmic side of the transporter [15] The affinities of these two sites for glucose are very different Pseudosubstrates such as 3FGlc, or inhibitors such as the epoxide 1d would interact preferentially with the glucose low-affinity site To the contrary, the C-1 epoxypropyl analogue 3a might react in the high-affinity site The inactivation data presented here, and in a previous study [42], indicate that Cys421 is accessible to reactive, membrane-impermeable reagents, such as analogues 1a,b, from the periplasmic side (a) EIIGlc, EIIMan and the mannitol transporter, EIIMtl display biphasic phosphorylation kinetics towards their natural substrates, indicating that activity in vitro is the sum of contributions from two independent sites (Fig 6), one of high affinity and low turnover, the second one of low affinity and high turnover [15,41] There exist, however, pseudosubstrates, for which EII displays Michaelis–Mentenlike kinetics 3-Deoxy-3-fluoro-D-glucose (3FGlc), for instance, preferentially if not exclusively binds to the lowaffinity site of EIIGlc, as deduced from the same Ki measured for a C-6 aldehyde analogue of Glc as inhibitor of Glc and 3FGlc phosphorylation by EIIGlc [15] The newly synthesized C-1 epoxide 3a is the first and so far only analogue that preferentially binds to the Glc high-affinity site of EIIGlc The Km and Vmax of EIIGlc for 3a are lower than for Glc, and 3a is a strong competitive inhibitor of uptake (b) The high-affinity periplasmic site and the low-affinity cytoplasmic site of the transporter recognize different features of the substrate Of six analogues that reversibly inhibited EIIGlc at the cytoplasmic site (in membrane preparations), only one, 3a, also inhibited uptake by intact cells (Fig 6) A comparison of structure and reactivity between the six analogues suggests that inhibitors of uptake that bind to the periplasmic site of the protein must have a free OH-6, whereas inhibitors of phosphorylation that bind to the cytoplasmic site may or may not have one Thus, the C-1 epoxide 3a with a free OH-6 was a potent inhibitor of uptake, whereas the most potent inhibitor of nonvectorial phosphorylation, the C-6 epoxide 1d, was a comparatively weak inhibitor of uptake Like 1d, two glucose-6-aldehyde analogues have recently been shown to display a similar preference for the low-affinity site [15] (c) Substrate protection is commonly used to confirm the specificity of an active-site labelling reaction Addition of Glc, however, did not protect but sensitized EIIGlc for inactivation (Table 3) This could indicate that binding of Glc to one site increases the reactivity of a second site Also pointing in this direction is the second-order or biphasic shape of the inactivation curve of EIIGlc by 1a (see residuals in Fig 3) This may indicate that binding of a first molecule of 1a to the EIIGlc dimer does not inactivate, but increases the reactivity of, Cys421 towards a second molecule Alternatively, biphasic inactivation by 1a (with two inactivation rates differing by a factor of 10) may originate from the different accessibility of the two cysteines of the EIIGlc dimer for the glucose analogues For comparison, inactivation by iodoacetamide fits better to an exponential function (Fig 3) as expected of a small nonspecific reagent with equal access to both Cys Whatever the cause, sensitization by Glc cannot be the (trivial) effect of Glc-induced dephosphorylation/deprotection of Cys421, because EIIGlc in membrane preparations is already dephosphorylated [6], as indicated by the complete inactivation induced by iodoacetamide The bromoacetyl derivatives 1a and 1c also inactivated Glc uptake by starved cells Being modified at OH-6, 1a and 1c were neither substrates nor competitive inhibitors of uptake (see Fig 4) That they nevertheless inactivated EIIGlc suggests that the reactive Cys421 must be directly accessible from the periplasmic side of the membrane and that accessibility is increased in the presence of Glc Because this same effect was not observed with EIIMan-expressing cells, nonspecific effects on essential PTS components other that EIIGlc can be excluded What cannot be excluded is that dephosphorylation and/or catalytic turnover of EIIGlc, rather than binding of Glc, enhanced the reactivity of Cys421 As Cys421 is the only invariant cysteine in homologous transporters and also the only essential cysteine for IICBGlc activity [39], it must be the reactive one and accessible from the periplasm Our results confirm experiments of Robillard et al [42], who demonstrated that EIIGlc-dependent uptake can be inactivated by membraneimpermeable thiol reagents, and, on the basis of this, concluded 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Robillard, G.T (1993) Steady state kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the Escherichia coli phosphoenolpyruvate- dependent phosphotransferase system J Biol Chem 268, 17844– 17849 42 Robillard, G.T & Beechey, R.B (1986) Evidence for the existence of a channel in the glucose-specific carrier EIIGlc of the Salmonella typhimurium phosphoenolpyruvate-dependent phosphotransferase system Biochemistry 25, 1346–1354 SUPPLEMENTARY MATERIAL The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/ EJB3197/ EJB3197sm.htm Table S1 1H, 13C NMR and mass spectrometry (ESI) data for the synthetic intermediates 5, 7, 10 and 18  ... recently introduced for the preparation of C-6 aldehyde derivatives of Glc [15] The key step is the use of Collins reagent for the selective oxidation of the primary trimethylsilyl ether of the. .. RESULTS Synthesis of inhibitors The synthesis and characterization of compounds 1b, 1d, 1f, 1g and 3b (Scheme 1) is reported for the first time, and the epoxypropyl derivative 3a is prepared by a... Weber, G (1986) The oxidation of primary trimethylsilyl ethers to aldehydes: a selective conversion of a primary hydroxy group into an aldehyde group in the presence of a secondary hydroxy group J