Synthesis of phospho
enol
pyruvate (PEP) analogues and evaluation
as inhibitors of PEP-utilizing enzymes
Luis Fernando Garcı
´
a-Alles and Bernhard Erni
Departement fu
¨
r Chemie und Biochemie, Universita
¨
t Bern, Switzerland
The synthesis of 10 new phosphoenolpyruvate (PEP)
analogues with modifications in the phosphate and the
carboxylate function is described. Included are two potential
irreversible inhibitors of PEP-utilizing enzymes. One incor-
porates a reactive chloromethylphosphonate function
replacing the phosphate group of PEP. The second contains
a chloromethyl group substituting for the carboxylate
function of PEP. An improved procedure for the prepar-
ation of the known (Z)- and (E)-3-chloro-PEP is also given.
The isomers were obtained as a 4 : 1 mixture, resolved by
anion-exchange chromatography after the last reaction step.
The stereochemistry of the two isomers was unequivocally
assigned from the
3
J
H-C
coupling constants between the
carboxylate carbons and the vinyl protons.
All of these and other known PEP-analogues were tested

functionalized molecule that plays a central role in metabo-
lism. It is not only important because of its high phosphate
group-transfer potential (DG ¼ )61.9 kJÆmol
)1
), but also
because it is a versatile C
3-
synthon in C–C, C–P and C–O
bond-formation reactions [1]. Representative examples of
the first function are the synthesis of ATP catalysed by
pyruvate kinase, and the transport with concomitant
phosphorylation of carbohydrates across the bacterial
membrane, mediated by the PEP:sugar phosphotransferase
system (PTS) [2]. Examples of the second function are the
fixation of CO
2
in plants (mediated by PEP carboxylase) [3],
the generation of natural phosphonates (PEP mutase) [4],
the first step in peptidoglycan cell-wall biosynthesis (cata-
lysed by UDP-GlcNAc enolpyruvyl transferase) and the
biosynthesis of aromatic amino acids (3-deoxy-
D
-arabino-
heptulosonate-7-phosphate synthase and 5-enolpyruvyl-
shikimate-3-phosphate synthase) [1].
Because of its pivotal role in metabolism, PEP has been
the subject of extensive chemical modification. Most of the
pseudosubstrates or competitive inhibitors discovered so far
differed from PEP by the presence of substitutions distal to
the phosphate group (position C-3, similar to compounds

a Alles. Departement fu
¨
r Chemie
und Biochemie. Universita
¨
t Bern. Freiestrasse 3. CH-3012 Bern,
Switzerland.
Fax: + 41 31/631 48 87, Tel.: + 41 31/631 37 92,
E-mail:
Abbreviations: PEP, phosphoenolpyruvate; PTS, phosphoenolpyru-
vate:sugar phosphotransferase system; FC, flash chromatography;
HRMS, high resolution mass spectrometry.
(Received 20 February 2002, revised 23 April 2002,
accepted 15 May 2002)
Eur. J. Biochem. 269, 3226–3236 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02995.x
PEP carboxylase, and enolase. Compounds 2a–e, 2g–i and
3c are new, and for that reason their synthesis and
characterization is also reported, as well as an improved
method for the preparation of the 3-Cl-PEP analogues 1c
and 1d. The last two isomers and the chlorinated analogues
2g and 3c are candidates for the irreversible inactivation of
PEP-utilizing enzymes.
MATERIALS AND METHODS
Enzyme I, and the rest of components from the PTS were
expressed and purified as previously described [19]. Pyruvate
kinase from rabbit muscle (2000 UÆmL
)1
),
L
-lactate dehy-

phosphate from Aldrich. PEP (cyclohexylammonium salt)
and NADH (disodium salt) were from Sigma. Solvents were
usually of the highest purity commercially available. Ben-
zene was dried by continuous refluxing over and distillation
from sodium. Fluka silica gel 60/230-400 mesh was used in
column chromatography purification. Ion-exchange chro-
matography was carried out using Dowex 50W X-8 (50–100
mesh) from Fluka and Sephadex DEAE A-25 column from
Pharmacia. Deuterated solvents were purchased from
Armar AG (D
2
O, CD
3
OD) and Fluka (CDCl
3
).
Characterization of the PEP-analogues 1–3, butyrate is
given in Table 1. (Z)-3-F-PEP (1b)and(Z)-phosphoenol-
butyrate [1e (Z)-3-Me-PEP] were a generous gift of
R. L. Somerville (Department of Biochemistry, Purdue
University, West Lafayette, IN, USA). They were contam-
inated with around 6% of their E-isomers, as judged
from their
1
H-NMR spectra. Phospho-
D
,
L
-lactic acid (1f)
was obtained via condensation of methyl-

Ethyl 3,3-dichloropyruvate (5a) was prepared by stirring
a mixture of fresh ethyl pyruvate (2.9 g, 25 mmol), sulfuryl
chloride (4.1 mL, 50 mmol), and p-toluenesulfonic acid
dihydrate (0.24 g, 1.25 mmol) at 70 °C. Extra sulfuryl
chloride (50 mmol) was added after 4 and 8 h of reaction.
The reaction was continued for a total of 24 h. Excess
sulfuryl chloride was removed by distillation and water
(10 mL) was added. The reaction mixture was extracted
with diethyl ether (3 · 15 mL), the organic layer dried over
anhydrous magnesium sulfate, and the solvent evacuated.
Silica gel flash chromatography (FC) (hexanes/ethyl acetate
Scheme 1.
Table 1.
1
H-NMR spectral data (D
2
O, noninterchangeable signals) of
analogues 1–3. Products as cyclohexylammonium salts, except 1c, 1d,
2b (triethylammonium), 3a (potassium salt) and 3b (acid form). Signals
due to cyclohexylamonium: d ¼ 3.11 p.p.m. (m, 1H), 1.94 (m, 2H),
1.76 (m, 2H), 1.62 (m, 1H), 1.30 (m, 5H) and triethylammonium:
d ¼ 3.17 p.p.m. (q, 6 H), 1.05 (t, 9H).
Product
d (p.p.m.) (multiplicity, J in Hz)
Vinyl protons R
1
,R
2
,R
3

5% of the oxidized form
also present: 4.56 (s), 3.41 (s). These signals disappear upon addi-
tion of dithiothreitol.
Ó FEBS 2002 Inhibitors of phosphoenolpyruvate-utilizing enzymes (Eur. J. Biochem. 269) 3227
7 : 3, v/v) furnished ethyl dichloropyruvate: 1.8 g, 40%.
1
H-NMR (CDCl
3
) d:5.97(1H,s,CHCl
2
), 4.38 (2H, q,
J ¼ 7.0 Hz, CH
2
O),1.36(3H,t,J ¼ 7.0 Hz, CH
3
).
Synthesis of the enolphosphates 1c,d and 2a-i
Step (1): Perkow reaction. Ten millimoles of ethyl
dichloropyruvate (5a)orthea-haloketones (4a–e)was
added dropwise to a flask containing 10 mmol (1.18 mL) of
trimethyl phosphite (20 mmol for the preparation of 6d)at
0–10 °C. Violent bubbling took place in some cases. After
addition, the ice-bath was removed and the reaction was
allowed to proceed at the temperature indicated below until
31
P-NMR indicated the complete disappearance of trimeth-
yl phosphite (typically overnight). Small amounts of
trimethyl phosphite were eliminated in vacuo (0.1 mbar) at
room temperature. Details: 6a: reaction at room tempera-
ture, purification by FC (hexanes/ethyl acetate, 2 : 3, v/v),

cribed by McKenna et al. was employed [24]. Trimethyl-
silyl bromide (2 mmol, 0.27 mL) was slowly added to a
flask containing 1 mmol of compound 6a–f or 7a,kept
under argon at 0–4 °C. 4 mmol of trimethylsilyl bromide
were used in the reaction with compound 6d. The mixture
was stirred for 1 h and then for an additional 1 h at room
temperature. After evaporation of excess trimethylsilyl
bromide at high vacuum, 2 mmol of cyclohexylamine in
15 mL of methanol/ether (1 : 5, v/v) were added. The
white solid was collected by filtration and washed with
3 · 8mL of ether. 2a: dicyclohexylammonium salt,
0.28 g, 70%. 2c: dicyclohexylammonium salt, 0.35 g,
89%. 2e: dicyclohexylammonium salt, 0.38 g, 97%. 2f:
tricyclohexylammonium salt, 0.58 g, 61% [25,26]. 2g:
dicyclohexylammonium salt, 0.33 g, 89%. 2h:dic-
yclohexylammonium salt, 0.25 g, 62%. 8a: dicyclohexyl-
ammonium salt, 0.30 g, 71%.
Step (3): Hydrolysis of carboxylic acid ester
groups. Compounds 2b, 2d and 2i were prepared from
2a, 2c and 2h, respectively. Compound 2b was obtained by
addition of 5 molar equvalents of KOH (1
M
) to the residue
obtained after evaporation of excess trimethylsilyl bromide
in the previous step. Hydrolysis was allowed to proceed for
3–4 min. The aqueous solution was passed through a
DowexWX-8column(H
+
-form) and the acidic fractions
were pooled and neutralized with 2 mmol of cyclohexyl-

M
HCl (final pH value ¼ 6.0). The two isomers were
separated following the procedure of Poyner et al. with
modifications [27]. The mixture was diluted with 300 mL
deionized water and slowly loaded at 4 °C to a Sephadex
DEAE A-25 column (30 g, Cl
–
form), which was then eluted
with a KCl gradient (2 mLÆmin
)1
, 10 mL per fraction,
0.15
M
to 0.35
M
in 475 min). The compounds were detected
at 254 nm. Product 1c started to elute at 0.19
M
,whereas1d
appeared at 0.27
M
KCl. The corresponding fractions were
pooled and diluted three times with deionized water. They
were loaded on a second Sephadex DEAE A-25 column
(HCO
3
–
form) and eluted with 2 mLÆmin
)1
tryethylammo-

J ¼ 3.7 Hz, CH
2
¼ C), 4.09 (2H, d, J ¼ 10.7 Hz, CH
2
Cl);
31
P-NMR (CDCl
3
) d: +26.2. The product 3c was obtained
after addition of 10 to 5 mL of ice-cold H
2
Oand
neutralization with 0.65 mL of cyclohexylamine. The solu-
tion was lyophilized and the product recovered by filtration
after triturating with 25 mL MeOH/ether, 1 : 4, v/v. 3c:
dicyclohexylammonium salt, 0.61 g, 15%.
Stability of PEP analogues 1–3
Most of PEP-derivatives 1–3 were stable over months
when stored as 250 m
M
solutions at pH ¼ 7.0–7.3 and
)20 °C. However, compounds 2b, 2d and 2i decomposed
under these conditions. Periodical inspection by
31
P-NMR revealed a continuous increase of the inorganic
phosphate signal (+1.96 p.p.m. at pH 7.1 in double-
distilled H
2
O).
Competitive inhibition enzyme assays

,1lLof
membrane extract, 1 m
MD
-glucose, 0.1 units
D
-glucose-
6-phosphate dehydrogenase, 1 m
M
NADP
+
,50m
M
Hepes
pH ¼ 7.5, 2.5 m
M
dithiothreitol and 2.5 m
M
NaF. Either
5m
M
MgCl
2
or 1 m
M
MnCl
2
were also present.
Pyruvate kinase activity was determined in a coupled
assay with
L

or 1 m
M
MnCl
2
.
Enolase inhibition by 1b–d (0–200 l
M
), 1f and 2f (with
Mn
2+
) was directly monitored as the increase of absorption
at 235 nm due to the formation of the conjugated C–C
double bond of PEP from
D
-2-phosphoglyceric acid [29].
Reversible inhibition with the rest of compounds was
assayed by coupling PEP formation with NADH consump-
tioninthepresenceofpyruvatekinaseand
L
-lactate
dehydrogenase [27]. The experiments were carried out in the
presence of 0.04 UÆmL
)1
of enolase and 5 m
M
MgCl
2
or
0.15 UÆmL
)1

of 1c,d,or5m
M
of 2g, 3a and 3c. Aliquots
(15–20 lL) were withdrawn at time intervals and diluted in
cold quenching buffer (285–130 lL) containing 1 m
M
PEP
or
D
-2-phosphoglyceric acid in the case of enolase. The
residual enzymatic activity was determined under the
conditions of the IC
50
assays, after addition of the enzyme
to a fresh mixture of the rest of components, 1 m
M
PEP or
D
-2-phosphoglyceric acid, and 5 m
M
MgCl
2
.
RESULTS
Preparation of the PEP-analogues 2a-i
The synthesis has been based in the Perkow reaction
(Scheme 2) [31]. The commercially available a-haloketones
4a–e were reacted with trimethyl phosphite, giving the
enolphosphate dimethyl esters 6a–e,inmostcasesin
quantitative yields. The thioester 6f was prepared from the

hexanes/AcOEt, 3 : 2, v/v]. The free carboxylate and
hydroxyl groups probably promote nucleophilic displace-
ments on the postulated phosphonium intermediate of the
Perkow reaction [31], thereby precluding the elimination of
methyl chloride. This course of the reaction is indicated by
the isolation of product 6a (R
2
¼ CH
2
CO
2
Me) from the
reaction between 4f and trimethyl phosphite. Therefore 2b
and 2d,aswellas2i were prepared by alkaline hydrolysis of
the esters 2a, 2c and 2h, respectively. However, 2a was
stable to hydrolysis at pH 12 and the reaction had to be
carried out under more harsh conditions (1
M
KOH). As a
consequence, small amounts of side-products were formed,
as shown by
1
H-NMR, and 2b had to be purified by anion-
exchange chromatography.
Ó FEBS 2002 Inhibitors of phosphoenolpyruvate-utilizing enzymes (Eur. J. Biochem. 269) 3229
Synthesis of potential irreversible inhibitors
Only a few irreversible inhibitors of PEP-utilizing enzymes
are described in the literature. Two examples are the
antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic
acid], which targets UDP-GlcNAc enolpyruvyl transferase

[28]. We instead decided to use the ethyl ester 5a, because it
afforded a 1 : 4 mixture of the (E)- and (Z)-isomers 7a,
therefore allowing the simultaneous preparation of the two
isomers 1c and 1d. Besides, in our hands, the compound 1c
obtained following the described procedure was contamin-
ated with around 5% PEP, which could not be removed.
This contamination probably derives from the presence of
small amounts of 3-chloropyruvic acid mixed with the
3,3-dichloropyruvic acid prepared following the reported
procedure.
The derivative 2g and the ethyl esters 8a (Z/E mixture)
were obtained after treatment of 6e and 7a with trimeth-
ylsilyl bromide and methanolysis. Finally, the ethyl ester
group of 8a was hydrolysed under basic conditions, and the
Z-andE-isomers were separated by anion-exchange
chromatography. Compounds 1c and 1d could be obtained
in this way at the same time and in higher than 99% purity
(Fig. 1A,B).
The analogue 3c carries a chloromethylphosphonate
group instead of the phosphate present in PEP. This
functionality can react with nucleophiles located in the
active-site of PEP-utilizing proteins. 3c might be particularly
suited to label residues which are transiently phosphorylated
in the course of the catalytic cycle, for instance, the active-
site histidines of enzyme I of the PTS [2], phosphoenolpyru-
vate synthase, and pyruvate phosphate dikinase, or the
presumed active-site aspartic acid residue of phos-
phoenolpyruvate mutase [36].
The synthesis of the chloromethylphosphonate 3c was
accomplishedasdepictedinScheme3.Thestrategywas

3
OD without decoupling.
3230 L. F. Garcı
´
a-Alles and B. Erni (Eur. J. Biochem. 269) Ó FEBS 2002
based on the formation of the mixed cyclic anhydride 10,
which was expected to be readily hydrolysable to furnish 3c.
Similar five-membered ring phosphates are known to be
exceptionally susceptible to nucleophilic attacks [37], and
have been used as strong phosphorylating agents. An
analogous cyclic acyl phosphate is probably formed during
the intramolecular carboxylate-catalysed hydrolysis of PEP
phosphate esters [38], and was also proposed as an
explanation to the
18
O distribution pattern observed when
PEPisheatedinacidicH
18
2
O[39].
Compound 10 was prepared by a method used for the
preparation of similar structures [40]. The moisture-sensitive
trimethylsilyl 2-trimethylsilyloxypropenoate (9) prepared
from pyruvic acid [22], was reacted with chloromethylphos-
phonic acid dichloride.
31
P-NMR of the crude reaction
mixture revealed three major signals appearing upfield of the
chloromethylphosphonic acid signal. The cyclic acyl phos-
phate 10 could be isolated by distillation and was partially

1
H) for each isomer has now been measured. It is
known that, without exception, the coupling constant
between two nuclei substituted directly on the carbons of
a carbon–carbon double bond is stronger when they are in
the trans rather than the cis orientation [41].
3
J
HC
was
determined in two steps: (a) the phosphorus-carbon coup-
ling constant (HOO
13
C-C-O-
31
P) was measured in the
13
C-
NMR
1
H spin decoupled spectrum of the pure isomer
(Fig. 1C,D); and (b) the additional coupling, due to the
vinyl proton, was obtained from the coupled spectra
(Fig. 1E,F). The compound with the strongest
1
H-
13
C
coupling constant (
3

,
L
-lactic acid (1f) were screened as inhibitors of (a) enzyme I
of the PTS from E. coli; (b) rabbit muscle pyruvate kinase;
(c) maize PEP carboxylase; and (d) yeast enolase. Because
the selection of the metal cofactor required by many PEP-
dependent enzymes has been reported to influence the
inhibition results in some cases, the assays have been
performed with Mg
2+
-andMn
2+
-activated enzymes.
Derivatives 1b, 1c, 1e, 1f, 2f, 3a and 3b were used previously
to study some of these enzymes. They have been included in
the present work, for comparison, and to complete the data
for the four enzymes. However, due to the number of assays
to be performed IC
50
values were calculated. The results are
presented in Table 2. The inhibition type and the value of
the inhibition constant (K
i
) have been determined only in
some representative cases.
Inhibition of enzyme I. The bacterial PTS catalyses uptake
with simultaneous phosphorylation of the carbohydrates [2].
The PTS is a group transfer pathway: a phosphoryl group
derived from PEP is transferred sequentially along a series of
proteins to the sugar molecule. Enzyme I is the protein at the

is the replacement of the phosphate-bridging oxygen by a
CH
2
moiety, is completely inactive, suggesting that this
oxygen participates in hydrogen bonds with the enzyme or
in the coordination to the metal cofactor. Similar results
have been obtained with compound 3b as inhibitor of PEP
mutase [36], and pyruvate kinase [6].
Inhibition of pyruvate kinase. This enzyme catalyses the
regeneration of ATP from ADP and PEP, in the last step of
glycolysis. Due to its physiological relevance, pyruvate
kinase is one of the best studied enzymes and many PEP
analogues have been used with it [5–7,16,18,25,28,29,33,42].
Compounds 1–3 were tested as inhibitors of the reaction
betweenADPandPEPcatalysedbypyruvatekinase.
Pyruvic acid is one of the products of this reaction.
Therefore, activity was measured by coupling the formation
of pyruvate with its NADH-dependent reduction to
L
-lactate, a process catalysed by
L
-lactate dehydrogenase.
Compounds 1b,c potently inhibit phosphotransfer from
PEP to ADP, in accordance with their published inhibition
constants (K
i
): 57 n
M
for 1b [5], and 39 n
M

4
plants,
where it concentrates CO
2
before it enters the Calvin cycle.
Inhibition was studied by measuring the rate of oxalac-
etate formation from PEP in the presence of increasing
concentrations of compounds 1–3. Activity was detected in
a coupled assay with NADH/malate dehydrogenase. All
compounds were first checked as pseudosubstrates, in order
to verify incompatibilities with the inhibition studies. The
activity detected with the known substrates of PEP
carboxylase 1b and 1c was very low [15,28,30]. With the
rest of compounds no activity could be detected, indicating
that either they are not substrates of PEP carboxylase, or the
products formed are not substrates of malate dehydroge-
nase. PEP carboxylase inhibition was then measured. Again
the most potent inhibitors with respect to PEP were those
modified at C-3. Measured IC
50
values are well correlated
with the reported K
i
:85l
M
for 1b [30], 63 l
M
for 1c [28],
and 18 l
M

L
-isomer of
Table 2. Half-inhibitory concentrations (IC
50
) and half inactivation times (t
50
) of PEP-utilizing enzymes with analogues 1–3. IC
50
values (given in m
M
)
were obtained using 0.1 m
M
PEP, in the presence of 5 m
M
MgCl
2
or 1 m
M
MnCl
2
. t
50
values (given in min) were measured at 30 °C with 0.5 m
M
1c,d or 5 m
M
2g, 3a and 3c, in the presence of 5 m
M
MgCl

Mg
2+
Mn
2+
t
50
a
Mg
2+
Mn
2+
t
50
a
PEP 0.2 [52] – 0.03 [5] 0.02 [5] 0.8 [28] 0.3 [28] 0.05 [5] 0.08 [5]
Modified in vinyl region
1b 0.8 NM 1 · 10
)4
NM 0.07 NM 3 · 10
-4
NM
1c 0.9 NM 0.7 5 · 10
)5
NM ND 0.02 NM ND 0.25 NM ND
1d >10 NM  60 0.12 NM ND 0.4 NM ND 0.25 NM ND
1e >10 NM 0.15 NM 0.04 NM 0.04 NM
1f >10 5  7 0.16 0.5 0.08 1.9 5
Modification of the carboxylic group
2a >10 >10 >10 >10 25%
c

b
Using 0.1 m
MD
-2-phosphoglyceric acid, in the presence of 5 m
M
MgCl
2
or 2 m
M
MnCl
2
.
c
Enhancement of the activity observed upon addition of the compound. The percentage of increase of activity achieved with 5 m
M
of compound is indicated.
d
In presence of 5 m
M
MgCl
2
. Extrapolated from inhibition observed after 2 h incubation.
3232 L. F. Garcı
´
a-Alles and B. Erni (Eur. J. Biochem. 269) Ó FEBS 2002
phospholactate was reported to shift from 100 to 1 l
M
when
Mg
2+

isoenergetic [45]. Several PEP-analogues have been
employed in the study of enolase [5–7,16,46,47]. Two
compounds (Z)-3-F-PEP (1b)anda-(dihydroxyphosphinyl-
methyl)acrylate (3b) function as alternative substrates [6]. In
the case of (Z)-3-F-PEP the product of the reaction is the
enol of tartronate semialdehyde phosphate, a potent
reversible inhibitor of the enzyme. This compound is
formed after OH
–
attack at C-3, followed of F
–
elimination.
Enolase also catalyses the formation of tartronate semial-
dehyde phosphate from (Z)-3-Cl-PEP (1c) [27]. Neverthe-
less, these compounds display a far lower catalytic efficiency
than PEP, and for that reason it is still possible to study
them as reversible inhibitors.
Inhibition of enolase was followed in two ways. In most
of cases the formation of PEP from
D
-2-phosphoglyceric
acid was coupled to the pyruvate kinase/
L
-lactate dehy-
drogenase assay. Obviously, this methodology was not
applied with compounds 1b–e (also 1f and 2f when Mn
2+
was present) as they are good inhibitors of pyruvate kinase.
In these cases the formation of PEP was directly monitored
at 235 nm, in the presence of variable amounts of the

, Fig. 2D). Preserving the negative
charge of the carboxylate group of PEP seems to be essential
for recognition by enolase. It is likely that such a negative
charge is required for binding to one of the two divalent ions
present in the active site of enolase [48]. Finally, in the case
of enolase the metal selected did not affect the inhibition
results as markedly. Only with the compound 3b a strong
enhancement of inhibition was observed in the presence of
Mn
2+
. Under such conditions this analog noncompetitively
inhibits enolase with a K
i
of 6 l
M
(not shown).
Enzyme inactivation studies
To screen for irreversible/suicide inhibition, the target
enzymes were first incubated at 30 °C under turnover
conditions with the PEP analogues 1c,d, 2g and 3c in the
presence of Mg
2+
, and were then assayed for residual
catalytic activity with their natural substrates. Inactivation
of PEP carboxylase and enolase was also studied in the
presence of Mn
2+
. Enzyme I and PEP carboxylase were
also treated with 3a, as this compound might transfer the
sulfuryl group to a catalytic residue, thereby blocking the

vated pyruvate kinase by 0 l
M
(squares), 11 l
M
(circles), 33 l
M
(tri-
angles) and 100 l
M
(stars) compound 2f. (C) Inhibition of Mn
2+
-
activated PEP carboxylase in the presence of 0 m
M
(squares), 1 m
M
(circles), 3 m
M
(triangles) and 9 m
M
(stars) compound 3c. (D) Inhi-
bition of Mn
2+
-activated enolase by 0 m
M
(squares), 0.33 m
M
(cir-
cles), 1 m
M

¼ K
m
(1 + [I]/K
i
).
Ó FEBS 2002 Inhibitors of phosphoenolpyruvate-utilizing enzymes (Eur. J. Biochem. 269) 3233
Pyruvate kinase was not irreversibly inhibited by any of
the PEP analogues 1c,d, 2g or the chloromethylphospho-
nate 3c. Incubations were prolonged for up to 2 h at 30 °C
without significant effect. Slow inactivation of PEP
carboxylase was induced by compound 2g (25% of activity
loss after 2 h). Incubation with the 3-Cl-PEP isomers 1c and
1d for the same time inactivated PEP carboxylase by less
than 10%. It was therefore not possible to reproduce the
results obtained when the enzyme was incubated at 25 °C
with the analogue 1c inthepresenceofMn
2+
(reported
t
1/2
¼ 5 h) [28]. In the case of enolase no inactivation was
observed with compound 3c. Compounds 1c,d and 2g were
also tested, in spite of the fact that they were not expected to
behave as mechanism-based inhibitors, as the enzyme does
not catalyse dephosphorylation reactions. They were not
inhibitory.
DISCUSSION
The data presented in this work, in combination with
multiple studies presented previously with these and other
PEP-utilizing enzymes, indicate that the most active

previous results. However, some observations are of special
interest. For instance, the remarkable difference observed
between the Z-andE-isomers of 3-Cl-PEP as inhibitors: the
Z-isomer is three to four orders of magnitude stronger than
the E-isomer. A much smaller difference has been observed
between the two isomers of phosphoenolbutyrate, a com-
pound presenting a more voluminous substitution than
chlorine: 7.1 l
M
K
i
for (Z)-phosphoenolbutyrate (1e)vs.
49.5 l
M
for its E-isomer [29]. Consequently, the data
measured with the 3-Cl-PEP isomers cannot be justified
on the basis of steric arguments. Other electronic factors
must contribute differently with each isomer, to establish the
interactions with the enzyme.
From the enzymes studied in this work, PEP carboxylase
has been found to be the most tolerant to modifications on
the structure of PEP. In concrete, it is interesting to call the
attention to the results obtained with 2b, 2h and 2i.The
carboxymethyl, acetylsufanylmethyl and mercaptomethyl
functions of these analogues are either considerably bulkier
than the carboxylate group of PEP or electronically very
different. Therefore they are not expected to occupy the
pocket that PEP carboxylase uses for the carboxylate group
of PEP. A bicarbonate binding pocket is also present in this
enzyme. Consequently, these derivatives might exert inhibi-

´
a-Alles and B. Erni (Eur. J. Biochem. 269) Ó FEBS 2002
that is known to be present in close proximity to the
methylene group of PEP [50].
Interestingly, some of the PEP derivatives modified at the
carboxylic position did not inhibit but instead stimulated
the activity of PEP carboxylase, especially in the presence of
Mg
2+
. This effect might be due to binding of these
compounds to the glucose-6-phosphate allosteric site of
the enzyme, similarly to what has been observed with
fosfomycin by Mu´ jica-Jime
´
nez et al.[51].Inthatstudy,the
metal-free form of fosfomycin was proposed to compete
with free PEP for the enzyme’s allosteric site. Similarly,
complex formation with Mg
2+
is probably precluded in the
stimulatory compounds 2a,d,e,g, because neutral chemical
functions are replacing the coordinating carboxylate group
of PEP in these molecules. In agreement with this propo-
sition, the compounds presenting negatively charged groups
in that position, 2b and 2f, did not enhance PEP carboxylase
activity.
ACKNOWLEDGEMENTS
We are indebted to the Swiss National Science Foundation (grant
31–45838.95) and the Secretarı
´

8. Hoving, H., Nowak, T. & Robillard, G.T. (1983) Escherichia coli
phosphoenolpyruvate-dependent phosphotransferase system:
stereospecificity of proton transfer in the phosphorylation of
enzyme I from (Z)-phosphoenolbutyrate. Biochemistry 22,
2832–2838.
9. Kim, D.H., Tucker-Kellogg, G.W., Lees, W.J. & Walsh, C.T.
(1996) Analysis of fluoromethyl group chirality establishes a
common stereochemical course for the enolpyruvyl transfers cat-
alyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl trans-
ferase. Biochemistry 35, 5435–5440.
10. Sundaram, A.K. & Woodard, R.W. (2000) Probing the Stereo-
chemistry of E. coli 3-deoxy-
D
-arabino-heptulosonate 7-phosphate
synthase (phenylalanine-sensitive)-catalyzed synthesis of KDO
8-P analogues. J. Org. Chem. 65, 5891–5897.
11. Adlersberg, M., Dayan, J., Bondinell, W.E. & Sprinson, D.B.
(1977) Stereochemical studies of the pyruvate kinase reaction with
(Z)- and (E)-phosphoenol-alpha-ketobutyrate. Biochemistry 16,
4382–4387.
12. Dotson, G.D., Nanjappan, P., Reily, M.D. & Woodard, R.W.
(1993) Stereochemistry of 3-deoxyoctulosonate 8-phosphate syn-
thase. Biochemistry 32, 12392–12397.
13.Reed,G.H.,Poyner,R.R.,Larsen,T.M.,Wedekind,J.E.&
Rayment, I. (1996) Structural and mechanistic studies of enolase.
Curr. Opin. Struct. Biol. 6, 736–743.
14. Hwang, S.H. & Nowak, T. (1986) Stereochemistry of phosphoe-
nolpyruvate carboxylation catalyzed by phosphoenolpyruvate
carboxykinase. Biochemistry 25, 5590–5595.
15. Janc, J.W., Urbauer, J.L., O’Leary, M.H. & Cleland, W.W. (1992)

24. McKenna, C.E., Higa, M.T., Cheung, N.H. & McKenna, M.C.
(1977) The facile dealkylation of phosphonic acid dialkyl esters by
bromotrimethylsilane. Tetrahedron Lett. 155–158.
25. Bearne, S.L. & Kluger, R. (1992) Phosphoenol acetylpho-
sphonates: substrate analogs as inhibitors of phosphoenolpyruvate
enzymes. Bioorg. Chem. 20, 135–147.
26. Benenson, Y., Belakhov, V. & Baasov, T. (1996) 1-(Dihydroxy-
phosphynyl) vinyl phosphate: the phosphonate analog of phos-
phoenolpyruvate is a pH-dependent substrate of Kdo8P synthase.
Bioorg. Med. Chem. Lett. 6, 2901–2906.
27. Poyner, R.R., Laughlin, L.T., Sowa, G.A. & Reed, G.H. (1996)
Toward identification of acid/base catalysts in the active site of
enolase: comparison of the properties of K345A, E168Q, and
E211Q variants. Biochemistry 35, 1692–1699.
28. Liu, J., Peliska, J.A. & O’Leary, M.H. (1990) Synthesis and study
of (Z)-3-chlorophosphoenolpyruvate. Arch. Biochem. Biophys.
277, 143–148.
29. Duffy, T.H., Saz, H.J. & Nowak, T. (1982) Stereospecificity of
(E)- and (Z)-phosphoenol-alpha-ketobutyrate with chicken liver
phosphoenolpyruvate carboxykinase and related phospho-
enolpyruvate-utilizing enzymes. Biochemistry 21, 132–139.
30. Dı
´
az, E., O’Laughlin, J.T. & O’Leary, M.H. (1988) Reaction of
phosphoenolpyruvate carboxylase with (Z)-3-bromophos-
Ó FEBS 2002 Inhibitors of phosphoenolpyruvate-utilizing enzymes (Eur. J. Biochem. 269) 3235
phoenolpyruvate and (Z)-3-fluorophosphoenolpyruvate. Bio-
chemistry 27, 1336–1341.
31. Lichtenthaler, F.W. (1961) Chemistry and properties of enol
phosphates. Chem. Rev. 61, 607–649.

41. Bovey, F.A. (1969) Nuclear Magnetic Resonance Spectroscopy.
Academic Press, New York, NY.
42. Wirsching, P. & O’Leary, M.H. (1988) 1-Carboxyallenyl phos-
phate, an allenic analogue of phosphoenolpyruvate. Biochemistry
27, 1355–1360.
43. Gonzalez, D.H. & Andreo, C.S. (1986) Phosphoenolpyruvate
carboxylase from maize leaves. Studies using beta-methylated
phosphoenolpyruvate analogs as inhibitors and substrates. Z.
Naturforsch. C. Biosci. 41, 1004–1010.
44. Cohn, M., Pearson, J.E., O’Connell, E.L. & Rose, I.A. (1970)
Nuclear magnetic resonance assignment of the vinyl hydrogens of
phosphoenolpyruvate. Stereochemistry of the enolase reaction. J.
Am. Chem. Soc. 92, 4095–4098.
45. Burbaum, J.J. & Knowles, J.R. (1989) Internal thermodynamics
of enzymes determined by equilibrium quench: values of Kint for
enolase and creatine kinase. Biochemistry 28, 9306–9317.
46. Stubbe, J. & Abeles, R.H. (1980) Mechanism of action of enolase:
effect of the beta-hydroxy group on the rate of dissociation of the
alpha-carbon-hydrogen bond. Biochemistry 19, 5505–5512.
47. Appelbaum, J. & Stubbe, J.A. (1975) Enolase catalyzed beta,
gamma-alpha,beta isomerization of 2-phospho-3-butenoic acid to
(Z)-phosphoenol-alpha-ketobutyrate. Biochemistry 14, 3908–3912.
48. Larsen, T.M., Wedekind, J.E., Rayment, I. & Reed, G.H. (1996)
A carboxylate oxygen of the substrate bridges the magnesium ions
at the active site of enolase: structure of the yeast enzyme com-
plexed with the equilibrium mixture of 2-phosphoglycerate and
phosphoenolpyruvate at 1.8 A
˚
resolution. Biochemistry 35, 4349–
4358.

13
C-NMR and mass spectrometry data
of analogues 1-3.
Table S2.
1
H- and
31
P-NMR data of derivatives 6a–f, 7a
and 8a.
3236 L. F. Garcı
´
a-Alles and B. Erni (Eur. J. Biochem. 269) Ó FEBS 2002