The glucose-specific carrier of the Escherichia coli phosphotransferase system Synthesis of selective inhibitors and inactivation studies Luis Fernando Garcı´a-Alles, Vera Navdaeva, Simo
Trang 1The glucose-specific carrier of the Escherichia coli phosphotransferase system
Synthesis of selective inhibitors and inactivation studies
Luis Fernando Garcı´a-Alles, Vera Navdaeva, Simon Haenni and Bernhard Erni
Departement fu¨r Chemie und Biochemie, Universita¨t 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
phospho-transferase system (PTS) 2¢,3¢-Epoxypropyl b-D
-glucopyr-anoside (3a) is an inhibitor and also a pseudosubstrate Five
analogues are inhibitors of nonvectorial Glc phosphorylation
by EIIGlcbut not pseudosubstrates They are selective for
EIIGlcas demonstrated by comparison with EIIMan, another
Glc-specific but structurally different transporter 3a is the
only analogue that inhibits EIIGlcby binding to the
high-affinity 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-D
-gluco-heptopyranoside (1d), preferentially interacts with the
low-affinity cytoplasmic site but only weakly inhibits Glc
uptake Binding and/or phosphorylation from the
cyto-plasmic side of EIIGlcis more permissive than sugar binding
and/or translocation of substrates via the periplasmic site EIIGlcis rapidly inactivated by the 6-O-bromoacetyl esters of methyl a-D-glucopyranoside (1a) and methyl a-D -manno-pyranoside (1c), methyl 6-deoxy-6-isothiocyanato-a-D -glucopyranoside (1e), b-D-glucopyranosyl isothiocyanate (3c) and b-D-glucopyranosyl phenyl isothiocyanate (3d) Phosphorylation of EIIGlcprotects, indicating that inacti-vation occurs by alkylation of Cys421 Glc does not protect, but sensitizes EIIGlcfor 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 EIIGlcbecomes transiently accessible to substrate analogues on the periplasmic side of the transporter Keywords: binding site; carbohydrate chemistry; cysteine; glucose transporter; irreversible inhibitor
Escherichia coli has two transporters for glucose, EIIGlc
(IIAGlc-IICBGlc) [1] and EIIMan(IIABMan-IICMan-IIDMan)
[2,3], which mediate uptake concomitant with
phosphory-lation of their substrates The immediate source of
high-energy 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 phos-phorylated at His, whereas IIB domains are phosphos-phorylated
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 do 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 IICBGlcsubunits co-operate in so
Correspondence to L F Garcı´a Alles, Departement fu¨r Chemie und
Biochemie, Universita¨t Bern, Freiestrasse 3, CH-3012 Bern,
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;
IC 50 , half inhibitory concentration; FC, flash chromatography.
(Received 9 June 2002, revised 16 August 2002,
accepted 21 August 2002)
Trang 2far 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
glu-cose 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
EIIGlcor EIIMan These experiments served to study binding
to EII from the periplasmic space
M A T E R I A L S A N D M E T H O D S
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 IICBGlcwith
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 30C, using the coupled assay with L-lactate dehydrogenase and NADH Final assay concentrations were: 0.5 lMEI, 1 lMHPr, 15 lMIIAGlc(or 0.5 lM IIABMan) and 0.0013 lLÆlL)1 membrane extract Other conditions were as described in [15] Typical back-ground activities of 2 lmolÆmin)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 Glc6Pwas detected in these experiments using Glc6Pdehydrogenase (1 UÆmL)1) and 1 mMNADP [21]
Fitting of kinetic data usingDYNAFIT
This software is available free of charge at http://www biokin.com [22] The kinetic constants were estimated as reported [15]
Inactivation ofnonvectorial phosphorylation
A 10-lL sample of inhibitor in buffer A [50 mM Hepes,
pH 7.5, 4 mM dithiothreitol, 5 mM MgCl2, 1 mgÆmL)1 BSA, 0.5 mgÆmL)1egg yolk lecithin (Sigma)] was preincu-bated for 10 min at 30C, and then added to 40–60 lL purified IICBGlcin buffer A (final concentration: 10–3 lM) Then 10-lL aliquots were withdrawn at intervals and diluted into 290 lL buffer A at 4C The diluted IICBGlc samples were assayed for in vitro PTS activity using the
D-Glc6Pdehydrogenase assay The final concentrations in the activity assay were 0.08–0.04 lMIICBGlcand 2 mMGlc
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- -[14C]glucose ([14C]2dGlc) by starved cells
Scheme 1.
Trang 3expressing EIIManwas 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 coliK12 ZSC112LDG cells expressing either EIIGlcor
EIIMan were prepared as described previously [15] To
0.65 mL cell suspension in M9 medium (0.2–0.1 gÆmL)1) at
room temperature were added 26 lL of a stock solution of
the irreversible inhibitor (0.5–0.025M), with or without Glc
(0.25M) Aliquots (0.15 mL) were withdrawn at the
indi-cated time points and diluted into ice-cold M9 medium
(0.85 mL) The cells were collected by centrifugation and
resuspended in 1 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-6-isothiocyanato-a-D-glucopyranoside (1e) [24], and b-D
-glucopyranosyl isothiocyanate (3c) [25] were prepared by
the reported procedures Methyl 6-O-chloroacetyl-a-D
-glucopyranoside (1b) was prepared like 1a using
chloroace-tyl chloride instead of bromoacechloroace-tyl bromide The yield was
56% after flash chromatography (FC) (ethyl
acetate/meth-anol, 96 : 4, v/v):1H NMR (CD3OD) d: 4.66 (1H, d, J¼
3.7 Hz, H1), 4.47 (1H, dd, J¼ 11.8, 2.2 Hz), 4.31 (1H, dd,
J¼ 11.8, 6.3 Hz), 4.24 (2H, s, CH2Cl), 3.73 (1H, m), 3.61
(1H, dd, J¼ 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
-allopyr-anoside (2a) and methyl 3,4-anhydro-a-D
-galactopyrano-side (2b) were obtained by desilylation of 1 mmol of the
6-O-[dimethyl-(1,1,2-trimethylpropyl)silyl]-protected forms
[26] with CsF (3 mmol) in dimethylformamide (50 mL) at
110C, for 30 min Evaporation in vacuo of
dimethylform-amide, 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-D
-glucoheptopyranoside (1d)
Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside (4) was
obtained from methyl a-D-glucopyranoside, following
con-ventional sugar transformations [28] A solution of 4 (2.6 g,
5.6 mmol) in 12 mL dichloromethane was added dropwise
to a suspension of activated powdered 4 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 min 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-tri-O-benzyl-a-D-glucohexodialdo-1,5-pyranoside (5) [29] The
aldehyde 5 (1 g, 2.1 mmol, in 5 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
cooling bath was removed and stirring was continued for 1.5 h The reaction was stopped at 0C 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 8 : 2, v/v) gave 0.69 g (70%) methyl 6,7-dideoxy-2,3,4-tri-O-benzyl-a-D -gluco-hept-6-enopyranoside (6) [31] A solution of compound 6 (0.2 g, 0.43 mmol) in 5 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 0.12Maqueous 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 7 (0.12 g, 0.25 mmol) in 6 mL 4.4% formic acid in methanol was added to a suspension of 0.3 g palladium black in 8 mL 4.4% formic acid in methanol [32] After 30 min, the catalyst was removed by filtration through celite, and washing with methanol (2· 5 mL) After eva-poration 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-6-enopyranosiduronic acid (1g) and its methyl ester (1f) Methyl 2,3,4-tris-O-(trimethylsilyl)-a-D -gluco-hexodialdo-1,5-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, 1 mmol) in
5 mL dry dichloroethane After 2 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 Com-pound 10 (0.1 g, 0.21 mmol) was desilylated by stirring for
2 h at room temperature with methanol (2 mL) and K2CO3 (2 mg) After evaporation 54 mg 1f (100%) was obtained :
1H NMR (CD3OD) d: 7.29 (1H, dd, J¼ 15.8, 4.4 Hz, H6), 6.31 (1H, dd, J¼ 15.8, 1.8 Hz, H7), 4.93 (1H, d, J ¼ 4.0 Hz, H1), 4.13 (1H, ddd, J¼ 9.9, 4.4, 1.5 Hz, H5), 3.84 (1H, dd,
J¼ 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, J¼ 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, 0.1Msolution in water) was effected at pH 12 (1MKOH) for 2 h The derivative 1g was obtained as a potassium salt in quantitative yield:1H NMR (D2O, pD 7) d: 6.51 (1H, dd, J¼ 15.8, 7.0 Hz, H6), 6.11 (1H, dd, J¼ 15.8, 1.1 Hz, H7), 4.80 (1H, d, J¼ 3.7 Hz, H1, overlapped with water signal), 4.14 (1H, dd, J¼ 9.6, 7.0 Hz, H5), 3.70–3.58 (2H, m), 3.41 (3H, s,
CH3O), 3.33 (1H, dd, J¼ 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
Trang 42 mL dichloroethane was treated with freshly prepared
dimethyldioxirane in acetone (3 mL, 0.3 mmol) [34]
After 1 h, the ice bath was removed, and
dimethyldioxi-rane (2 mL) was added after 2 and 4 h The reaction was
continued overnight Solvent was removed by
evapor-ation, and the resulting epoxypropyl (13) (93 mg) was
deacetylated as described [35], to furnish 3a as a 6 : 4
diastereomeric mixture:1H NMR (CD3OD) d: 4.52 (0.4H,
d, J¼ 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 5 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
concen-trated The residue was chromatographed (hexane/diethyl
ether, 7 : 3, v/v) giving 2.3 g (52%) of 16 The epoxide 17
was then prepared from 16 by reaction with
dimethyldioxi-rane, as described [36] The derivative 17 (0.24 g,
0.55 mmol) was treated with 5 mL racemic glycidol at
room temperature After 2 h reaction, the excess glycidol
was removed under vacuum, and the residue was
chroma-tographed with diethyl ether, giving 0.13 g of 14 [37] The
epoxide 14 (50 mg, 0.1 mmol) was debenzylated in 30 min
by following the same method as for 1d 23 mg of a 1 : 1
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 : 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, 9 : 1, v/v) resulted
in 73 mg (88%) compound 3b:1H NMR (CD3OD) d: 5.73
(1H, d, J¼ 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%)
R E S U L T S
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 1 were synthesized following
described procedures, with modifications as specified in
Materials and Methods All compounds were characterized
by1H-NMR and13C-NMR spectroscopy and by
electro-spray MS
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 4 [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 4 to aldehyde with pyridinium chlo-rochromate, followed by Wittig methylenation at C-6, epoxidation of the newly created double bond of 6 with 3-chloroperoxybenzoic acid, and removal of the protecting benzyl groups present in 7 by catalytic transfer hydrogen-ation 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 carboxy-lic 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 trimethyl-silyl ether of the fully trimethyl-silylated monosaccharide 8 to an aldehyde (step ii) The resulting 2,3,4-tris-trimethylsilylated derivative 9 was then condensed with methyl triphenyl-phosphoranylidene 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-H7¼ 15.8 Hz) Removal of the trimethylsilyl groups afforded the methyl ester 1f This compound was hydro-lyzed 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 4 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
Scheme 2.
Trang 5at 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 con-verted 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 -gluco-pyranoside 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
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 phosphoenolpyru-vate) 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 Kmof EIIGlcfor 3a
is 28 lM, which is comparable to the 60 lM value determined in a parallel experiment for Glc (Table 1) Vmax
of EIIGlcfor 3a was 14.9 lMÆmin)1which is only three times slower than for Glc (40 lMÆmin)1) Compound 3a is highly selective for EIIGlcand is not phosphorylated by EIIMan The C-1 chloroacetyl derivative 3b induced a slow con-sumption of NADH in the presence ofL-lactate dehydro-genase, but also formation of NADPH in the presence of Glc6Pdehydrogenase 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 3 : 2 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
Compounds 1a)3d were assayed in vitro as inhibitors of Glc phosphorylation by the two PTS transporters The concen-tration of the glucose analogues was varied between 0 and
5 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 Phosphoryl-ation of Glc was measured with the Glc6Pdehydrogenase-coupled assay Representative data for three compounds are shown in Fig 1, and IC50of all compounds are listed in Table 2 Without exception, EIIGlc was more strongly inhibited than EIIMan The C-6 epoxide 1d was the strongest inhibitor It inhibited EIIGlcwith an IC50of 0.07 mM, but had almost no effect on EIIMan This result was confirmed
Scheme 3.
Scheme 4.
Trang 6with [14C]aMGlc as substrate and direct detection of
[14C]aMGlc6P(results not shown) The second best
C-6-modified analogues, Glc (1a),
bromoacetyl-Man (1c), and isothiocyano-Glc (1e) had a 10 times higher
IC50than 1d The epimeric bromoacetyl derivatives 1a and
1c both inhibited EIIGlc, although EIIGlcstrongly
discrimi-nates 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
pseudosub-strate) and the chloroacetyl 3b had an IC50of 1 mM The
remaining analogues with bulky and rigid substituents had
IC50> 2.4 mMor 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
phosphoryl-ation was measured at four different concentrphosphoryl-ations of 3a
and 1d, and the results were plotted in the Eadie–Hofstee
form (Fig 2) In the absence of an inhibitor, EIIGlc displayed biphasic kinetics (Fig 2, solid symbols), consis-tent 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 2 were fitted to the two-active-site model that was recently introduced to explain kinetic data collected with several glucose analogues as pseudo-substrates 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/KS2¼ 0.04 at the low-affinity site, and KI1/KS1¼ 5 at the high-affinity site The curved shape of the plot and the KI/KSratios 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),
Fig 1 Inhibition of nonvectorial phosphorylation Relative rate of Glc
phosphorylation by membranes containing EII Glc (solid symbols) and
EII Man (open symbols) in the presence of inhibitors 1d (squares), 1e
(triangles) and 3a (circles) The IC 50 values obtained from these and
similar plots are listed in Table 2 [Glc] ¼ 0.5 m M Glc
phosphoryla-tion was detected with the -Glc6Pdehydrogenase assay.
Table 1 Kinetic constants of EII Glc and EII Man foranalogues of D -glucose Phosphorylation was measured at 30 C with the L -lactate dehydro-genase-coupled assay Kinetic constants were derived from a best fit to a Michaelis–Menten hyperbola NS, No saturation observed.
Substrate
V maxa
(l M Æmin)1)
K m
(l M )
V max /K ma
( · 10 3 min)1)
V maxa
(l M Æmin)1)
K m
(l M )
V max /K ma
( · 10 3 min)1)
a
Using 0.0013 lLÆlL)1membrane extract Concentrations of other PTS components are indicated in Materials and methods.bUsing freshly purified compound The reaction is also detected with the D -Glc6Pdehydrogenase assay.
Table 2 Compounds 1a)3d as inhibitors of D -glucose phosphorylation Phosphorylation of D -Glc (0.5 m M ) was measured using the D -Glc6P dehydrogenase-coupled assay at 30 C in the presence of 0–5 m M
concentrations of the inhibitors ND, No significant inhibition detec-ted Half inhibitory concentrations (IC 50 ) are given in m M Values in parentheses determined measuring inhibition of phosphorylation of [ 14 C]a-MGlc (0.5 m M ).
Inhibitor
IC 50
a Confirmed with the radioactivity-based assay.
Trang 7the corresponding ratios were KI2/KS2 ¼ 5 and KI1/
KS1¼ 0.7 These values and the transition of the Eadie–
Hofstee plot from the biphasic to a monophasic shape are
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 incuba-tion For instance, inactivation of EIIGlcby iodoacetamide was 50% in the presence of 0.5 mM, and almost complete in the presence of 4 mMdithiothreitol (results not shown) For this reason EIIGlc-containing membranes were always preincubated in the presence of 4 mMdithiothreitol 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 EIIGlcin the phosphorylated state (+ PEP) Aliquots were with-drawn 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 2 Reversible inhibition of EIIGlc Eadie–Hofstee plots of
non-vectorial phosphorylation of D -Glc (0–2 m M ) by membrane fractions
containing EIIGlc Phosphorylation was assayed in the presence of
inhibitors 1d [A, 0 l M (squares), 33.3 l M (triangles), 100 l M (circles)
and 300 l M (stars)] and 3a [B, 0 m M (squares), 0.33 m M (triangles),
1 m M (circles) and 3 m M (stars)] The lines represent the best global
least-squares fit of the data to a kinetic model of EII with two
inde-pendent 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)
K S1 ¼ 4 l M , k 1 ¼ 23 min)1, K I1 ¼ 19 l M , K S2 ¼ 190 l M ,
k 2 ¼ 39 min)1, K I2 ¼ 8 l M ; with 3a (B) K S1 ¼ 4 l M , k 1 ¼ 16 min)1,
K I1 ¼ 3 l M , K S2 ¼ 140 l M , k 2 ¼ 32 min)1, K I2 ¼ 700 l M K S1 and
K S2 are the dissociation constants of E1 and E2 for Glc, K I1 and K I2 the
dissociation constants for the inhibitor, and k 1 and k 2 are the
turn-over numbers DYNAFIT was used to fit the experimental data to the
theoretical model and in the subsequent simulations [22].
Table 3 Rates of inactivation of IICB Glc Incubation of purified IICBGlcwith the indicated concentration (m M ) 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.
k inact
a-Haloester analogues
Epoxides
Isothiocyanates
a,b-Unsaturated carboxylic acid derivatives
a
Incubation in the simultaneous presence of 10 m M Glc.bIncubation in the presence of 1.5 m M phosphoenolpyruvate, 0.5 l M E1, 0.5 l M
HPr, 1 l IIA Glc c Rate constants calculated by nonlinear fit to a second-order decay function: y ¼ A/(1 + k t) + residual.
Trang 8Representative 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 3 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 mMGlc
did not protect against inactivation To the contrary, the
presence of Glc slightly sensitized EIIGlcfor 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 EIIGlcconcentrations present during the
incuba-tion, can accept a phosphoryl group and thereby deprotect
Cys421
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 EIIGlcand EIIMan, respectively, and conse-quently 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 : 1 and the uptake of [14C]aMGlc via EIIGlcor 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 EIIGlconly, 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 bromo-acetyl-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 EIIGlcand EIIMan P reincu-bation with 20 mMC-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 bromoace-tyl-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 2 and 5 min 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 3 Irreversible inhibition of EIIGlc A membrane preparation
containing EIIGlcwas preincubated with 1 m M inhibitor 1a (circles),
iodoacetamide (stars) and bromoacetic acid (tickmarks) in the presence
of 4 m M 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).
Fig 4 Inhibition of sugaruptake by star ved cells EIIGlc-dependent uptake of [ 14 C]aMGlc (0.1 m M , black bars), and EII Man -dependent uptake of [14C]2dGlc (0.1 m M , grey bars) in the presence of the indicated inhibitors (1 m M ) DPTS, Background uptake by a strain lacking both EII Glc and EII Man 100% uptake corresponds to
25 nmolÆmin)1Æmg)1 dry weight of cells expressing EIIGlc, and
90 nmolÆmin)1Æmg)1cells expressing EIIMan.
Trang 9mannose epimer 1c (Fig 5A) whereas the opposite was true
for EIIMan(Fig 5B) This indicates that EIIGlcand 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, EIIGlcfor
inactivation (Fig 5A, grey bars) In the presence of Glc, the
rates of EIIGlcinactivation by 1a and 1c increased 18-fold
and 27-fold, respectively This effect of Glc is specific for
EIIGlcand 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 EIIGlcor EIIMan Cell growth in mineral medium supple-mented 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 (> 4 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
D I S C U S S I O N
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 pseudosub-strates, reversible and irreversible inhibitors of EIIGlcand EIIMan The C-1 epoxide analogue 3a was the only efficient pseudosubstrate of EIIGlcin 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 bromo-acetylglucose and bromoacetylmannose derivatives 1a,c irreversibly blocked in vitro phosphorylation and uptake
by starved cells The isothiocyanates only blocked in vitro phosphorylation by EIIGlcin membrane preparations, but not uptake The C-6 bromoacetyl derivatives and isothio-cyanates presumably reacted with the active-site residue Cys421 This cysteine transfers the phosphoryl group from the IIAGlcsubunit to the OH-6 of the substrate in a double-displacement 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 chemi-cally more reactive but unspecific iodoacetamide, (b) EIIGlc
is completely protected against inactivation if Cys421 is phosphorylated or converted into a disulfide before expo-sure to the alkylating analogues (results not shown), and (c) inactivation of EIIGlcis 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 con-firmed recent, insight into (a) the kinetic properties [15], (b) the selectivity, and (c) the conformational coupling of the EIIGlcactive sites
Fig 5 Glucose-sensitized inactivation of [14C]sugaruptake by starved
cells Cells expressing EIIGlc(A) or EII Man (B) were incubated for
2 min with and without inhibitors (10 m M ) in the absence (black bars)
and presence (grey bars) of 10 m M 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 4.
Table 4 Inactivation of sugaruptake by star ved cells by compounds
1a-3d Cells were treated with the indicated concentrations (m M ) of
inhibitor for 60 min at room temperature The rate of accumulation of
radioactive sugar in the pretreated cells was then measured Uptake
rates are in nmolÆmin)1Æmg)1dry weight of cells EIIGlcwas measured
using [ 14 C]aMGlc (0.1 m M , 6400 d.p.m.Ænmol)1) EII Man was
meas-ured using [ 14 C]2dGlc (0.1 m M , 5600 d.p.m.Ænmol)1).
Inhibitor Concn
Uptake rate
Trang 10(a) EIIGlc, EIIManand 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–Menten-like kinetics 3-Deoxy-3-fluoro-D-glucose (3FGlc), for
instance, preferentially if not exclusively binds to the
low-affinity site of EIIGlc, as deduced from the same Kimeasured
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
Kmand Vmaxof EIIGlcfor 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 EIIGlcby 1a (see residuals
in Fig 3) This may indicate that binding of a first molecule
of 1a to the EIIGlcdimer does not inactivate, but increases the reactivity of, Cys421 towards a second molecule Alternatively, biphasic inactivation by 1a (with two inacti-vation 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, inactiva-tion by iodoacetamide fits better to an exponential funcinactiva-tion (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 EIIGlccan 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 IICBGlcactivity [39], it must be the reactive one and accessible from the periplasm Our results confirm experi-ments of Robillard et al [42], who demonstrated that EIIGlc-dependent uptake can be inactivated by membrane-impermeable thiol reagents, and, on the basis of this, concluded that a reactive thiol group must be accessible from the periplasmic side
In conclusion, chemically reactive glucose analogues turned out to be instrumental in the characterization of EIIGlc as a dimeric transport protein with two mutually interacting binding sites containing an active-site cysteine that is accessible from both faces of the membrane The nature of the structural rearrangement for this alternating accessibility is now being examined with heterodimers between variants with mutations in the different domains
A C K N O W L E D G E M E N T S
This study was supported by grant 3100-063420 from the Swiss National Science Foundation.
R E F E R E N C E S
1 Bouma, C.L., Meadow, N.D., Stover, E.W & Roseman, S (1987) II-BGlc, a glucose receptor of the bacterial phosphotransferase system: molecular cloning of ptsG and purification of the receptor from an overproducing strain of Escherichia coli Proc Natl Acad Sci USA 84, 930–934.
2 Erni, B., Zanolari, B & Kocher, H.P (1987) The mannose permease of Escherichia coli consists of three different proteins Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA J Biol Chem 262, 5238–5247.
Fig 6 Proposed model for the IICBGlcdimerof EIIGlc IICBGlc
con-sists of a membrane-spanning C domain (grey) and the cytoplasmic
IIB domain (black) IICBGlcis phosphorylated at Cys421 by the
sol-uble IIA Glc subunit It is proposed that two nonvectorial
phosphory-lation sites are present at the cytoplasmic side of the transporter [15].
The affinities of these two sites for glucose are very different
Pseudo-substrates such as 3FGlc, or inhibitors such as the epoxide 1d would
interact preferentially with the glucose low-affinity site To the
con-trary, 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.