The 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 (EII
Glc
)ofthe
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 EII
Glc
but not pseudosubstrates. They are selective for
EII
Glc
as demonstrated by comparison with EII
Man

D
-
glucopyranoside (1e), b-
D
-glucopyranosyl isothiocyanate
(3c)andb-
D
-glucopyranosyl phenyl isothiocyanate (3d).
Phosphorylation of EII
Glc
protects, indicating that inacti-
vation occurs by alkylation of Cys421. Glc does not protect,
but sensitizes EII
Glc
for inactivation by 1e and 3d,whichis
interpreted as the effect of glucose-induced conformational
changes in the dimeric transporter. Glc also sensitizes EII
Glc
for inactivation by 1a and 1c of uptake by starved cells. This
indicates that Cys421 which is located on the cytoplasmic
domain of EII
Glc
becomes 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, EII
Glc
(IIA
Glc

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 EII
GlcNAc/Glc
[11]. EI, HPr and IIA are phos-
phorylated at His, whereas IIB domains are phosphorylated
at Cys421 in EII
Glc
and at His175 in EII
Man
.EII
Glc
is
specific for Glc, but EII
Man
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,
EII
Glc
and EII
Man
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

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)
Eur. J. Biochem. 269, 4969–4980 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03197.x
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 EII
Glc
and EII
Man
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 EII

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 IICB
Glc
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 EII
Glc
and EII
Man
, and purified
EII
Glc
, EI, HPr, IIA
Glc
and IIAB
Man
were prepared as
described in [15].
In vitro

determined by measuring the phosphorylation rate of
0.5 m
MD
-Glc, in the presence of 0–5 m
M
concentrations
of the inhibitors. Glc6P was detected in these experiments
using Glc6P dehydrogenase (1 UÆmL
)1
)and1m
M
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 m
M
Hepes,
pH 7.5, 4 m
M
dithiothreitol, 5 m
M
MgCl
2

-glucopyranoside ([
14
C]aMGlc)
by starved E. coli K12 ZSC112LDG cells expressing EII
Glc
or of 2-deoxy-
D
-[
14
C]glucose ([
14
C]2dGlc) by starved cells
Scheme 1.
4970 L. F. Garcı
´
a-Alles et al.(Eur. J. Biochem. 269) Ó FEBS 2002
expressing EII
Man
was assayed as described previously [15].
Transport rates were calculated from the amount of
[
14
C]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 EII
Glc
or
EII
Man

1
HNMR(CD
3
OD) 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, CH
2
Cl), 3.73 (1H, m), 3.61
(1H, dd, J ¼ 9.5, 8.8 Hz), 3.40 (3H, s. CH
3
O), 3.39 (1H,
overlapped), 3.30 (1H, m).
13
CNMR(CD
3
OD) 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
110 °C, for 30 min. Evaporation in vacuo of dimethylform-
amide, and FC (ethyl acetate/methanol, 96 : 4, v/v)

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 0 °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 MgSO
4
. 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 0.12
M
aqueous Na
2
S
2

+
, 100%).
Synthesis of methyl (6
E
)-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 K
2
CO
3
(2 mg). After evaporation 54 mg 1f (100%) was obtained :
1
HNMR(CD
3
OD) d:7.29(1H,dd,J ¼ 15.8,4.4Hz,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

O), 3.33 (1H, dd, J ¼ 9.6, 8.8, 1.1 Hz, H4);
13
CNMR
(D
2
O, 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].Anice-cooledsolutionof12 (90 mg, 0.23 mmol) in
Ó FEBS 2002 Inhibition of EII
Glc
(Eur. J. Biochem. 269) 4971
2 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)(93mg)was
deacetylated as described [35], to furnish 3a as a 6 : 4
diastereomeric mixture:
1
HNMR(CD

, 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.23mgofa1:1
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:
1
HNMR(CD
3
OD) d:5.73

-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 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
(
3
J

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 EII
Glc
and EII
Man
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 EII
Glc
. The apparent K
m
of EII
Glc
for 3a
is 28 l
M
, which is comparable to the 60 l
M
value
determined in a parallel experiment for Glc (Table 1). V
max

Man
. 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 EII
Glc
and EII
Man
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
5m
M
while the substrate,
D
-Glc, was kept constant at
0.5 m
M
. 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 Glc6P dehydrogenase-
coupled assay. Representative data for three compounds are
shown in Fig. 1, and IC
50
of all compounds are listed in

50
than 1d. The epimeric bromoacetyl derivatives 1a and
1c both inhibited EII
Glc
, although EII
Glc
strongly discrimi-
nates between Glc and Man. The chemically less reactive
chloroacetyl-Glc (1b) did not inhibit EII
Glc
. 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 IC
50
of 1 m
M
.The
remaining analogues with bulky and rigid substituents had
IC
50
>2.4m
M
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. EII
Glc
-dependent Glc phosphoryl-

I1
/K
S1
¼ 5 at the high-affinity site.
The curved shape of the plot and the K
I
/K
S
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
EII
Glc
. 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
D
-Glc6P dehydrogenase assay.

/K
m
a
( · 10
3
min
)1
)
V
max
a
(l
M
Æmin
)1
)
K
m
(l
M
)
V
max
/K
m
a
( · 10
3
min
)1

) are given in m
M
.Valuesin
parentheses determined measuring inhibition of phosphorylation of
[
14
C]a-MGlc (0.5 m
M
).
Inhibitor
IC
50
EII
Glc
EII
Man
1a 0.8 (2) ND
1b >5 ND
1c 0.8 (3) ND
1d 0.07
a
>5
1e 0.9 (2) ND
1f  5
a
ND
1g >5
a
ND
2a ND ND

: low for C-6,
high for the C-1 epoxides. They were determined at 0.5 m
M
Glc, at which concentration the high-turnover site (low
affinity) is saturated and therefore preponderant in catalysis.
Glucose analogues as irreversible inhibitors of EII
Glc
To assay for irreversible inhibition, membrane fractions
containing EII
Glc
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 EII
Glc
by iodoacetamide
was 50% in the presence of 0.5 m
M
, and almost complete in
the presence of 4 m
M
dithiothreitol (results not shown). For
this reason EII
Glc
-containing membranes were always
preincubated in the presence of 4 m
M
dithiothreitol. Three
conditions were assayed: (a) treatment with the inhibitor

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),
1m
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
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

2
1 0.61 ± 0.06 0.54 ± 0.09 < 0.001
BrAcOH 1 0.101 ± 0.004 – < 0.001
Epoxides
1d 60 0.007 ± 0.001 – –
2a 60 < 0.001 – –
2b 60 < 0.001 – –
3a 60 0.0038 ± 0.0004 – –
Isothiocyanates
1e 15 0.35 ± 0.04 0.7 ± 0.1 < 0.001
3c 15 0.54 ± 0.04 0.58 ± 0.06 0.4 ± 0.1
3d 5 0.55 ± 0.09 0.67 ± 0.06 < 0.001
a,b-Unsaturated carboxylic acid derivatives
1f 60 < 0.001 – –
1g 60 < 0.001 – –
a
Incubation in the simultaneous presence of 10 m
M
Glc.
b
Incubation in the presence of 1.5 m
M
phosphoenolpyruvate, 0.5 l
M
E1, 0.5 l
M
HPr, 1 l
M
IIA
Glc

Glc
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 EII
Glc
concentrations 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
[
14
C]sugar uptake by intact cells. The nonmetabolizable
[
14
C]aMG and [
14
C]2dGlc were used as substrates, instead
of [
14
C]glucose. These glucose analogues are selectively
transported via EII
Glc
and EII
Man
, respectively, and conse-
quently further guarantee that uptake is due to the studied

then the residual uptake activity was determined (see
Table 4). The C-6 bromoacetyl compounds 1a and 1c
completely blocked uptake by EII
Glc
and EII
Man
.Preincu-
bation with 20 m
M
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 bromoace-
tyl-Man (1c) was examined in more detail. Taking into
account that Glc appeared to sensitize rather than protect
EII
Glc
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 m
M
Glc. With short
incubation times, cells expressing EII
Glc
were inactivated
slightly faster by the glucose analogue 1a than by the
Fig. 3. Irreversible inhibition of EII
Glc
. A membrane preparation
containing EII

M
). DPTS, Background uptake by a strain
lacking both EII
Glc
and EII
Man
. 100% uptake corresponds to
25 nmolÆmin
)1
Æmg
)1
dry weight of cells expressing EII
Glc
,and
90 nmolÆmin
)1
Æmg
)1
cells expressing EII
Man
.
4976 L. F. Garcı
´
a-Alles et al.(Eur. J. Biochem. 269) Ó FEBS 2002
mannose epimer 1c (Fig. 5A) whereas the opposite was true
for EII
Man
(Fig. 5B). This indicates that EII
Glc
and EII

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 m
M
concentration required), 1d (> 4 m
M
), 1e
(> 0.2 m
M
), 3c and 3d (> 0.8 m
M
). 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 pseudosub-
strates, reversible and irreversible inhibitors of EII
Glc
and
EII
Man
. The C-1 epoxide analogue 3a was the only efficient

phosphorylated or converted into a disulfide before expo-
sure to the alkylating analogues (results not shown), and (c)
inactivation of EII
Glc
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 con-
firmed recent, insight into (a) the kinetic properties [15], (b)
the selectivity, and (c) the conformational coupling of the
EII
Glc
active sites.
Fig. 5. Glucose-sensitized inactivation of [
14
C]sugar uptake by starved
cells. Cells expressing EII
Glc
(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
MD
-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 sugar uptake by starved cells by compounds

EII
Glc
EII
Man
– – 23 ± 10 41 ± 17
1a 1 < 0.02 < 0.02
1c 1 < 0.02 < 0.02
1d 510±4–
20 4 ± 3 19 ± 3
1e 5 16±2 14±1
20 6 ± 4 8 ± 2
3a 517±9–
20 8 ± 5 17 ± 4
3d 5 20 ± 6 6.4 ± 0.8
20 5 ± 1 2 ± 1
Ó FEBS 2002 Inhibition of EII
Glc
(Eur. J. Biochem. 269) 4977
(a) EII
Glc
,EII
Man
and the mannitol transporter, EII
Mtl
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-

atthecytoplasmicsite(inmembrane
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 EII
Glc
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 EII
Glc
by 1a (see residuals
in Fig. 3). This may indicate that binding of a first molecule
of 1a to the EII
Glc
dimer does not inactivate, but increases
the reactivity of, Cys421 towards a second molecule.
Alternatively, biphasic inactivation by 1a (with two inacti-

,
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 IICB
Glc
activity [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
EII
Glc
-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
EII
Glc
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.
ACKNOWLEDGEMENTS
This study was supported by grant 3100-063420 from the Swiss
National Science Foundation.
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