Synthesis of phospho enol pyruvate PEP analogues and evaluationas inhibitors of PEP-utilizing enzymes Luis Fernando Garcı´a-Alles and Bernhard Erni Departement fu¨r Chemie und Biochemie,
Trang 1Synthesis 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 3JH-C coupling constants between the
carboxylate carbons and the vinyl protons
All of these and other known PEP-analogues were tested
as reversible and irreversible inhibitors of Mg2+- and Mn2+
-activated PEP-utilizing enzymes: enzyme Iof the
phos-phoenolpyruvate:sugar phosphotransferase system (PTS),
pyruvate kinase, PEP carboxylase and enolase Without exception, the most potent inhibitors were those with sub-stitution of a vinyl proton Modification of the phosphate and the carboxylate groups resulted in less effective com-pounds Enzyme Iwas the least tolerant to such modifica-tions Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with
Mn2+ For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I(t1/2¼ 0.7 min)
Keywords: phosphoenolpyruvate analogues; chemical synthesis; inhibition; irreversible inhibitor; PEP-utilizing enzymes
Phosphoenolpyruvate (PEP) is a small and highly
functionalized molecule that plays a central role in
metabo-lism It is not only important because of its high phosphate
group-transfer potential (DG¼)61.9 kJÆmol)1), but also
because it is a versatile C3-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 CO2in 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 1b–e, Scheme 1) [5–7] Some of these compounds turned out
to be crucial in mechanistic studies of PEP-utilizing enzymes, for instance in the establishment of the stereo-chemical course of enzymatic processes mediated by enzyme Iof the PTS [8], UDP-GlcNAc enolpyruvyl trans-ferase and 5-enolpyruvyl-shikimate-3-phosphate synthase [9], 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [10], pyruvate kinase [11], KDOP synthase [12], enolase [13], PEP carboxykinase [14], and PEP carboxylase [15]
A representative example is the study of UDP-GlcNAc enolpyruvyl transferase, and 5-enolpyruvyl-shikimate-3-phosphate synthase, with (Z)-F-PEP (1b), an analogue that allowed the isolation and characterization of stable fluoro analogues of the otherwise unstable tetrahedral intermediate of the normal reaction [1]
The carboxylic and the phosphate functionalities of PEP have been modified less frequently Several studies indicated that both groups might be essential to establish the correct substrate–active site contacts in pyruvate kinase and enolase [6] Important exceptions are phosphoenolthiopyruvate [16], thiophosphoenolpyruvate [17], and the remarkable case of sulfoenolpyruvate (3a) a substrate that transfers its sulfuryl group to ADP in the presence of pyruvate kinase [18] This paper presents the results obtained with 17 PEP analogues (Scheme 1) as inhibitors of the reactions catalysed by the enzyme Iof the PTS, pyruvate kinase,
Correspondence to L F Garcı´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: garcia@ibc.unibe.ch
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)
Trang 2PEP 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
M A T E R I A L S A N D M E T H O D S
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-drogenase from rabbit muscle (550 UÆmg)1), and
glucose-6-phosphate dehydrogenase from yeast (350 UÆmg)1) were
from Boehringer Mannheim Malate dehydrogenase from
porcine heart (2700 UÆmL)1) and PEP carboxylase from
maize (50 UÆmL)1) were from Fluka Enolase from bakers
yeast (500 UÆmL)1) was purchased from Sigma NADP
(sodium salt), pyruvic acid, D-2-phosphoglyceric acid
(sodium salt), bromoacetyl bromide (4d),
1,3-dichloro-2-propanone (4e), bromo and chloro trimethylsilane, trimethyl
phosphite, methylD,L-lactate, chloromethylphosphonic acid
dichloride and potassium thioacetate were from Fluka
4-Chloroacetoacetic acid methyl ester (4a) and
1-acetoxy-3-chloroacetone (4b) were from TCIAmerica Ethyl pyruvate,
3-bromo-1,1,1-trifluoroacetone (4c) and dimethyl
chloro-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 (D2O, CD3OD) and Fluka (CDCl3)
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 their1H-NMR spectra Phospho-D,L-lactic acid (1f)
was obtained via condensation of methyl-D,L-lactate and
dimethyl chlorophosphate [20], followed by phosphate ester
demethylation using trimethylsilyl bromide (step 2, see
below), and hydrolysis of the carboxylic methyl ester at
pH¼ 12.0 (step 3) Published methods were also followed for the synthesis of sulfoenolpyruvate (3a) [18], and a-(dihydroxyphosphonylmethyl) acrylic acid (3b) [6]
1H- and 13C-NMR spectra were recorded at 300.1 and 75.47 MHz, respectively Spectra in D2O were calibrated against sodium 3-(trimethyl)propane-1-sulfonate (external standard).31P-NMR at 81 MHz were calibrated against a 85% phosphoric acid external standard (d¼ 0.00 p.p.m.) Due to the pH dependence of phosphate and phosphonate chemical shifts, phosphorus data reported for the final products were acquired in double-distilled H2O at
pH¼ 7.1–7.4 Iodide (m/z 126.9045) and taurocholate (m/z 514.2839) were used as internal standards in negative mode high-resolution ESI-MS measurements
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 70C 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)
(dd, 7.4, 2.9, 3H) 1f 4.38 (p, 7.0), 1.34 (d, 7.0, 3H) –
2a 4.76 (d, 1.1), 4.42 (d, 0.7) 3.24 (s, 2H)
3.73 (s, 3H) 2b 4.77 (dd, 2.2, 1.8), 4.56 (t, 1.8) 3.20 (s, br, 2H) 2c 4.84 (s, br), 4.57 (s, br) 4.52 (s, br, 2H)
2.14 (s, 3H) 2d 4.64 (s, br), 4.44 (s, br) 3.91 (s, br, 2H)
2g 4.82 (s, br) a , 4.68 (s, br) 4.09 (s, br, 2H) 2h 4.66 (s, br), 4.49 (s, br) 3.57 (s, 2H)
2.35 (s, 3H) 2ib 4.61 (s, br), 4.48 (s, br) 3.16 (s, br, 2H) 3a 5.84 (d, 2.2), 5.58 (dd, 2.2) –
3b 6.29 (d, 5.7), 5.84 (d, 5.7) 2.86 (d, 22.3, 2H) 3c 5.54 (dd, 1.8, 3.7), 5.23
(dd, 4.1, 2.2)
3.57 (m, 2H)
a Partially overlapped with water signal.b5% of the oxidized form also present: 4.56 (s), 3.41 (s) These signals disappear upon addi-tion of dithiothreitol.
Trang 37 : 3, v/v) furnished ethyl dichloropyruvate: 1.8 g, 40%.
1H-NMR (CDCl3) d: 5.97 (1H, s, CHCl2), 4.38 (2H, q,
J¼ 7.0 Hz, CH2O), 1.36 (3H, t, J¼ 7.0 Hz, CH3)
Synthesis of the enolphosphates 1c,d and 2a-i
Step (1): Perkow reaction Ten millimoles of ethyl
dichloropyruvate (5a) or the a-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
0–10C 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
31P-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),
1.8 g, 80% 6b: reaction at room temperature, FC (hexanes/
ethyl acetate, 1 : 1, v/v), 1.45 g, 65% 6c: prepared following
Cherbuliez et al instructions [21], replacing the triethyl
phosphite for trimethyl phosphite, 2.2 g, 100% 6d:
pre-pared from bromoacetyl bromide (4d, R2¼ Br, X ¼ Br),
1 h reaction at 60C, 2.54 g, 100% [22] 6e: 2.0 g, 100%
[23] 7a: reaction at 70C for 1 h Purified by FC (hexanes/
ethyl acetate, 1 : 1, v/v): 1.5 g, 58% yield, 4 : 1 mixture of
the Z- and E-isomers
Preparation of the enolphosphate dimethyl ester 6f A
mixture of 6e (1 g, 5 mmol) and potassium thioacetate
(5.7 g, 5 mmol) was stirred into 5 mL of
dimethylforma-mide, at room temperature The reaction mixture was
sonicated periodically After 3 h, 10 mL of diethyl ether
were added, and the resulting suspension was passed
through a small path of silica gel, with hexanes/ethyl
acetate (1 : 1, v/v) as eluent The product fractions were
collected and the solvent removed.1H-NMR revealed the
presence of around 20% starting material, together with
the desired product To drive the reaction to completion
the mixture was subjected to two more reaction cycles,
adding consecutively 2 and 1 mmol of potassium
thioac-etate, until all starting material 6e had disappeared 6f:
0.53 g, 45%
Step (2): Removal of phosphate ester groups in 6a–f and
7a The simple and mild demethylation procedure
des-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 0–4C 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· 8 mL 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% 2 h:
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 2 h, respectively Compound 2b was obtained by addition of 5 molar equvalents of KOH (1M) 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 Dowex WX-8 column (H+-form) and the acidic fractions were pooled and neutralized with 2 mmol of cyclohexyl-amine The product was further purified by anion-exchange chromatography, following the procedure described for the separation of (Z)- and (E)-3-Cl-PEP (see below) It was detected after the first chromatography step at 220 nm Fifty-milliliter fractions were collected and lyophilized after the second chromatography, giving the
tristriethylammoni-um salt of 2b: 0.1 g, 20% yield
With compounds 2d and 2i, the cyclohexylammonium cations of 2c or 2h (1 mmol in 2–3 mL of deionized water) were first exchanged against Na+by loading on a Dowex XW-8 column (Na+form) The sodium salts were eluted with 3· 5 mL deionized water and adjusted to pH 12.0– 12.5 with 1 M KOH Around 3–4 mmol of KOH were usually added before reaction completion (1–2 h) The whole reaction volume was passed through the Doxew XW-8 column (4C, H-form), the eluate neutralized with cyclohexylamine (2 mmol, 0.23 mL) and then lyophilized 2d: dicyclohexylammonium salt, 0.30 g, 86% 2i: dicyclohexylammonium salt, 0.28 g, 75%
(Z)- and (E)-3-chlorophosphoenolpyruvate (1c,d) A portion (1.2 g; 2.8 mmol) of the 4 : 1 mixture of isomers 8a was hydrolysed similarly to compounds 2c and 2 h The solution was kept at pH 12.5 for 5 h and then neutralized with 1MHCl (final pH value¼ 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 4C 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, 0.15Mto 0.35Min 475 min) The compounds were detected
at 254 nm Product 1c started to elute at 0.19M, whereas 1d appeared at 0.27MKCl The corresponding fractions were pooled and diluted three times with deionized water They were loaded on a second Sephadex DEAE A-25 column (HCO3 form) and eluted with 2 mLÆmin)1 tryethylammo-nium bicarbonate (0.2Mto 1Min 475 min) The fractions containing the product were pooled and lyophilized Analytical HPLC (DEAE-60-7, Macherey–Nagel, condi-tions in legend to Fig 1) revealed that the isolated products were more than 99% pure 1c: ditriethylam-monium salt, 0.47 g, 42% [28] 1d: ditriethylamditriethylam-monium salt, 0.11 g, 10%
Chloromethylphosphonate 3c Trimethylsilyl 2-trimethylsilyloxypropenoate (9) was pre-pared as previously described [22]
Chloromethylphosphon-ic acid dChloromethylphosphon-ichloride (10 mmol, 1 mL) in 20 mL of dry benzene was added dropwise to a flask containing 2.3 g (10 mmol)
of 9 at 50C The reaction mixture was refluxed for 4 h Benzene was removed under vacuum, and the unstable cyclic acylphosphate 10 was Kugelrohr distilled at around
130C (0.1 mbar): 0.4 g, 22%,1H-NMR (CDCl), d: 5.82
Trang 4(1H, dd, J¼ 3.7, 1.8 Hz, CH2¼ C), 5.54 (1H, d,
J¼ 3.7 Hz, CH2¼ C), 4.09 (2H, d, J ¼ 10.7 Hz, CH2Cl);
31P-NMR (CDCl3) d: +26.2 The product 3c was obtained
after addition of 10 to 5 mL of ice-cold H2O and
neutralization with 0.65 mL of cyclohexylamine The
solu-tion was lyophilized and the product recovered by filtrasolu-tion
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 mM solutions at pH¼ 7.0–7.3 and
)20 C However, compounds 2b, 2d and 2i decomposed
under these conditions Periodical inspection by
31P-NMR revealed a continuous increase of the inorganic
phosphate signal (+1.96 p.p.m at pH 7.1 in
double-distilled H2O)
Competitive inhibition enzyme assays
Unless otherwise indicated, all experiments were performed
at 30C, in 96-well microtitre plates Progress curves were
recorded and the initial rates were calculated as the maximal
slope of the absorption curve obtained IC50 values were
measured using 0.1 mMPEP (0.1 mM D-2-phosphoglyceric
acid in the case of enolase) and in the presence of 0–5 mM
inhibitor at the enzyme and metal concentrations indicated
below
Enzyme Iactivity was measured by coupling the
formation of glucose-6-phosphate to its oxidation to
6-phosphoglucono-d-lactone This process is catalysed by
D-glucose-6-phosphate dehydrogenase and produces
NADPH, which can be monitored at 340 nm The reaction
conditions were as described (150 lL per well) [19]:
0.02 lM enzyme I, 1 lM HPr, 20 lM IIAGlc, 1 lL of
membrane extract, 1 mM D-glucose, 0.1 units D
-glucose-6-phosphate dehydrogenase, 1 mMNADP+, 50 mMHepes
pH¼ 7.5, 2.5 mMdithiothreitol and 2.5 mM NaF Either
5 mMMgCl2or 1 mMMnCl2were also present
Pyruvate kinase activity was determined in a coupled
assay with L-lactate dehydrogenase The initial rates of
formation of pyruvic acid released from PEP were
monit-ored by the decrease of absorption at 340 nm due to NADH
consumption, as described previously [5,29] The assays
were carried out in the presence of 0.015 UÆmL)1 of
pyruvate kinase and 5 mM MgCl2 or 0.05 UÆmL)1 of
pyruvate kinase and 1 mMMnCl2
PEP carboxylase activity was determined in a coupled
assay with malate dehydrogenase, as described previously
[30] The rate of formation of oxalacetic acid was
calculated from the rate of disappearance of NADH
Studies were conducted in the presence of 0.3 UÆmL)1of
PEP carboxylase and either 5 mM MgCl2 or 1 mM
MnCl2
Enolase inhibition by 1b–d (0–200 lM), 1f and 2f (with
Mn2+) was directly monitored as the increase of absorption
at 235 nm due to the formation of the conjugated C–C
double bond of PEP fromD-2-phosphoglyceric acid [29]
Reversible inhibition with the rest of compounds was
assayed by coupling PEP formation with NADH
consump-tion in the presence of pyruvate kinase and L-lactate
dehydrogenase [27] The experiments were carried out in the
presence of 0.04 UÆmL)1of enolase and 5 mM MgCl2 or 0.15 UÆmL)1of enolase and 2 mMMnCl2
Enzyme inactivation experiments The time-dependent inactivation assays were carried out under turnover conditions The enzymes (5 lM enzyme I,
3 UÆmL)1pyruvate kinase, 1.1 UÆmL)1PEP carboxylase or
2 UÆmL)1enolase) were preincubated for 10 min at 30C
in the presence of enough of the rest of components to maintain multiple turnovers (as indicated above) MgCl2 (5 mM) was present during the incubation (also 0.5 mM MnCl2 with PEP carboxylase and enolase) together with 0.5 mM of 1c,d, or 5 mM 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 mMPEP
or D-2-phosphoglyceric acid in the case of enolase The residual enzymatic activity was determined under the conditions of the IC50assays, after addition of the enzyme
to a fresh mixture of the rest of components, 1 mMPEP or
D-2-phosphoglyceric acid, and 5 mMMgCl2
R E S U L T S 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, in most cases in quantitative yields The thioester 6f was prepared from the 1-chloromethyl-vinyl derivative 6e, by nucleophilic displace-ment with potassium thioacetate Subsequent replacedisplace-ment
of the phosphate methyl ester for trimethylsilyl groups, by treating with trimethylsilyl bromide [24], and final methanolysis furnished 2a–i These compounds were purified by precipitating their cyclohexylammonium salts All attempts to synthesize 2b and 2d from the haloketones 4f (R2¼ CH2CO2H, X¼ Cl) and 4g (R2¼ CH2OH,
X¼ Cl), obtained after enzymatic hydrolysis of 4a and 4b, were unsuccessful [Note that 4a (5 mmol) was hydro-lysed with the lipase B from Candida antarctica (0.5 g) after
2 h at 37C in water-saturatedtBuOMe (50 mL) White needles of 4-chloro-3-oxo-butyric acid (4f) formed (62% yield) after removal of the enzyme by filtration, evaporation
of the solvent and recrystallization from hexanes/MeOH,
4 : 1, v/v 4b was hydrolyzed under the same conditions in the presence of Lypozyme 1-Chloro-3-hydroxy-propan-2-one (4g) was obtained in 74% yield after FC with 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 (R2¼ CH2CO2Me) from the reaction between 4f and trimethyl phosphite Therefore 2b and 2d, as well as 2i were prepared by alkaline hydrolysis of the esters 2a, 2c and 2 h, respectively However, 2a was stable to hydrolysis at pH 12 and the reaction had to be carried out under more harsh conditions (1MKOH) As a consequence, small amounts of side-products were formed,
as shown by1H-NMR, and 2b had to be purified by anion-exchange chromatography
Trang 5Synthesis 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
[32], and (Z)-3-bromo-phosphoenolpyruvate [(Z)-Br-PEP],
employed as a mechanism-based inhibitor of pyruvate kinase [33], pyruvate phosphate dikinase [5], and PEP carboxylase [30] We present here the preparation of four candidates for the irreversible inhibition of PEP-utilizing enzymes
The enolphosphates 1c,d, and 2g can be considered as potential suicide inhibitors They are nonreactive molecules but are transformed by enzyme-catalysed dephosphorylation into enolates, which in turn by protonation/tautomerization are converted to 3-chloropyruvic acid (in the case of 1c,d) or chloroacetone (from 2g) These a-halocarbonyl compounds can then react with nucleophilic amino-acid residues [34,35] Because they will be generated in the active site of the protein, the probability of labelling catalytically relevant residues, therefore inactivating the enzyme, is increased
These compounds were also synthesized via the Perkow reaction (Scheme 2) Ethyl 3,3-dichloropyruvate (5a) or commercially available 1,3-dichloroacetone (4e) were reac-ted with trimethyl phosphite The dimethyl enolphosphates 7a and 6e were obtained in excellent yields The preparation
of 6e by this route had been reported previously [23] On the other hand, the synthesis of the isomeric mixture 7a resembles the procedure proposed by Liu et al for the preparation of pure (Z)-3-chlorophosphoenolpyruvate (1c) from 3,3-dichloropyruvic acid (5b, R2¼ CO2H, X¼ Cl) [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- and E-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 Iof the PTS [2], phosphoenolpyru-vate synthase, and pyruphosphoenolpyru-vate phosphate dikinase, or the presumed active-site aspartic acid residue of phos-phoenolpyruvate mutase [36]
The synthesis of the chloromethylphosphonate 3c was accomplished as depicted in Scheme 3 The strategy was
Scheme 2.
Scheme 3.
Fig 1 Stereochemical assignment of (Z)-3-Cl-PEP (left) and
(E)-3-Cl-PEP (right) (A,B) Analytical anion-exchange HPLC of purified 1c
and 1d Chromatographic analysis was carried out in a DEAE-60-7
column [1 mLÆmin)1, 20 m M KH 2 PO 4 , pH ¼ 6.0, KCl (0 m M for
2 min to 360 m M in 16 min)] The effluent was monitored at 240 nm.
Retention times for each isomer are indicated (C,D) 1 H-decoupled
13
C-NMR spectra of 1c and 1d (ditriethylammonium salts) in CD 3 OD.
Only the carboxylate carbon region is shown (E,F)13C-NMR spectra
in CD OD without decoupling.
Trang 6based 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 the18O distribution pattern observed when
PEP is heated in acidic H18
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 31P-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
characterized by1H- and31P-NMR, in spite of its instability
As expected, hydrolysis of 10 produced compound 3c
Characterization of PEP analogues
Compounds 1–3 were characterized by1H (see Table 1),
13C- and 31P-NMR and mass spectrometry The
stereo-chemistry of compounds 1c and 1d could not be established
by comparison with published information, which disagree
in this respect The major product of the Perkow reaction
between 3,3-dichloropyruvic acid and trimethyl phosphite
was first reported by Liu et al to be the Z-isomer [28]
However, Poyner et al indicated that the dominant product
of the same reaction was the E-isomer [27] In view of this
contradiction the NMR coupling constant (3JHC) between
the carboxylic carbon atom and the vinyl proton (HOO13
C-C¼ C-1H) 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] 3JHC was
determined in two steps: (a) the phosphorus-carbon
coup-ling constant (HOO13C-C-O-31P) was measured in the13
C-NMR 1H 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 1H-13C
coupling constant (3JHC¼ 7.2 Hz, 3JPC¼ 5.7 Hz) was
assigned as the E-isomer and the compound with the
weakest coupling (3JHC¼ 1.6 Hz, 3JPC¼ 1.5 Hz) was
assigned as the Z-isomer Therefore (E)-Cl-PEP is the
compound presenting the vinyl proton signal (6.17 p.p.m.,
D2O, pH¼ 7.0) upfield from the vinyl signal of the
Z-isomer (6.72 p.p.m., D2O, pH¼ 7.1), in agreement with
Liu et al [28]
PEP analogues as reversible inhibitors of PEP-utilizing
enzymes
The compounds presented in Scheme 1 and phospho-D,
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 Mg2+- and Mn2+-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 IC50values were calculated The results are presented in Table 2 The inhibition type and the value of the inhibition constant (Ki) 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 Iis the protein at the top of this system It transfers the phosphoryl group from PEP to a phosphocarrier protein, HPr (Z)-phosphoenol-butyrate (1e) is the only analogue that has been used to study this enzyme It was employed to establish the stereochemistry of protonation of the released enolate [8] Before measuring inhibition, compounds 1–3 were checked as phosphoryl donors to enzyme Iin a glucose phosphotransferase assay Glucose 6-phosphate formation occurred with compounds 1b–e, but with one to three orders
of magnitude lower catalytic rates than with PEP (data not shown) All compounds were then tested as competitive inhibitors with respect to PEP As shown in Table 2, only the Z-isomers of 3-F-PEP (1b) and 3-Cl-PEP (1c) weakly inhibit enzyme I Inhibition by these compounds is com-petitive and the same Ki¼ 0.4 mM was calculated for the two compounds [Fig 2A, only shown for (Z)-3-F-PEP] Inhibition by analogues with a hydroxymethylene (2d) or a phosphonate group (2f) instead of the carboxylate of PEP, and byD,L-phospholactic acid (1f) was weak In contrast to the rest of enzymes shown in Table 2, no significant differences were observed when changing the metal present Interestingly, compound 3b, in which only difference to PEP
is the replacement of the phosphate-bridging oxygen by a
CH2 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 between ADP and PEP catalysed by pyruvate kinase 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 byL-lactate dehydrogenase Compounds 1b,c potently inhibit phosphotransfer from PEP to ADP, in accordance with their published inhibition constants (Ki): 57 nM for 1b [5], and 39 nM for 1c [28] Strikingly, however, E-Cl-PEP 1d is 2400-fold less inhibitory than its Z-isomer 1c
In a general sense, modification of the phosphate group
or the carboxylate function is counterproductive for binding
Trang 7to pyruvate kinase (Table 2) Nevertheless, a remarkable
dependence of inhibition on the metal employed is observed
Compound 2f had been described previously as not
interacting with Mg2+-activated pyruvate kinase [25] We
have found, however, that this compound becomes a strong
inhibitor when Mn2+is present Under such conditions, 2f
inhibits competitively pyruvate kinase with a Ki¼ 80 lM
(Fig 2B) This observation might be of relevance, for
instance, in efforts intended to cocrystallise pyruvate kinase
with a nonreactive PEP analogue and ADP Similar metal
dependence is observed with the racemic mixture of
compounds 1f and to a lesser extent with sulfoenolpyruvate,
3a, in agreement with published data [18]
Inhibition of PEP carboxylase This enzyme catalyses the
addition of bicarbonate to PEP to produce oxalacetate and
inorganic phosphate [3] PEP carboxylase is widely
distri-buted in plants It is particularly important in C4 plants,
where it concentrates CO2before 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 IC50values are well correlated with the reported Ki: 85 lMfor 1b [30], 63 lMfor 1c [28], and 18 lMfor 1e [43] The results also highlight a common feature of PEP-utilizing enzymes: the preference for Z- over E-isomers (compare IC50values measured for 1c and 1d)
In presence of Mg2+most of the PEP-derivatives with modifications of the carboxylic position do not inhibit and instead stimulate PEP carboxylase activity The effect is more pronounced in the presence of the trifluoromethyl and chloromethyl analogues 2e and 2g Besides, only com-pounds presenting neutral groups instead of the carboxylate function of PEP are stimulatory In contrast, none of the compounds that present negatively charged groups at this position, namely 2b or 2f, induce an increase in the activity
It is important to point out that the inhibition studies have been carried out at pH 7.5 Under similar conditions the compound 2f has previously been described to be a competitive inhibitor, with a Kiof 2.2 mM[25]
Similarly to pyruvate kinase, metal ion plays an import-ant role in PEP carboxylase inhibition Several compounds, e.g phospholactate (1f), become inhibitors in the presence
of Mn2+ The inhibition constant for the -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 2 K m values for PEP (m M ) are indicated; these values were taken from the indicated references NM, not measured; ND, no time dependent inactivation detected.
Comp
Modified in vinyl region
NM
Modification of the carboxylic group
Modified in the phosphate position
a
Incubation also in the presence of 0.5 m M MnCl 2 bUsing 0.1 m M D -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.
Trang 8phospholactate was reported to shift from 100 to 1 lMwhen
Mg2+was replaced by Mn2+[18] Five compounds, among
those modified at the carboxylic position, inhibit moderately
Mn2+-activated PEP carboxylase In presence of this metal,
inhibition by compounds 2b (not shown), 2h (not shown)
and 3c (Fig 2C) are competitive with Kivalues of 1.1, 1.2
and 3.9 mM, respectively Thus, PEP carboxylase is the only
enzyme shown in Table 2 that is able to interact with the
chloromethyl phosphonate 3c, arguing in favour of its
structural tolerance A second effect related to the metal
present is that only the compound 2g is now able to
stimulate the activity in the presence of Mn2+
Inhibition of enolase Enolase is a glycolytic enzyme that
catalyses the reversible elimination of water from
2-phospho-D-glycerate to form PEP The reaction proceeds
with anti stereochemistry [44], via a carbanion (enolate)
intermediate generated by abstraction of the C-2 proton of
D-2-phosphoglyceric acid [27] The reaction is nearly
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) and a-(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 Mn2+ 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 PEP-analogues
Compound 1b strongly inhibited enolase Other com-pounds, such as 1c–e and 3b were good competitors compared to D-2-phosphoglyceric acid Unlike the other enzymes assayed, enolase did not discriminate between the (Z)- and the (E)-3-Cl-PEP isomers Phospholactate 1f, the carboxymethyl analogue 2b and the phosphonate 2f displayed moderate inhibitory potencies The last analogue has been described to competitively inhibit enolase, with a
Kiof 2.2 mM[25] The same type of inhibition and similar inhibition constant value was measured for 2b in the presence of both Mg2+ (Ki¼ 2.0 mM, not shown) and
Mn2+ (Ki¼ 2.2 mM, 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
Mn2+ Under such conditions this analog noncompetitively inhibits enolase with a Kiof 6 lM(not shown)
Enzyme inactivation studies
To screen for irreversible/suicide inhibition, the target enzymes were first incubated at 30C under turnover conditions with the PEP analogues 1c,d, 2g and 3c in the presence of Mg2+, 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 Mn2+ Enzyme Iand PEP carboxylase were also treated with 3a, as this compound might transfer the sulfuryl group to a catalytic residue, thereby blocking the enzyme
Incubation of enzyme I with (Z)-3-Cl-PEP (1c) resulted
in a fast time-dependent inactivation (Fig 3) The time to half-inactivate the enzyme (t1/2) was 0.7 min Multiple turnover conditions were required, highlighting the suicidal nature of this inhibitor The E-isomer promoted a much slower irreversible inhibition (t1/2¼ 60 min) No inactiva-tion was detected with the rest of derivatives (Table 2) Details on the enzyme I/1c interaction have been recently reported [19]
Fig 2 Lineweaver–Burk plots of inhibition of PEP-utilizing enzymes in
the presence of PEP analogs 1–3 (A) Inhibition of Mg 2+ -activated
enzyme Iby 0 m M (squares), 0.12 m M (circles), 0.36 m M (triangles)
and 1.08 m M (stars) (Z)-3-F-PEP (1b) (B) Inhibition of Mn 2+
-acti-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 Mn2+
-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 Mn2+-activated enolase by 0 m M (squares), 0.33 m M
(cir-cles), 1 m M (triangles) and 3 m M (stars) analog 2b PEP concentrations
( D -2-phosphoglyceric acid with enolase) were varied between 0 and
2 m M in all cases except for (B) (0–0.5 m M ) Other conditions were as
indicated under Materials and methods for the calculation of IC 50
values The same data, plotted in the Michaelis–Menten form were
used to derive the K app
m for PEP or D -2-phosphoglyceric acid at each inhibitor concentration K i values were then calculated by linear
regression of K app
m values vs inhibitor concentration [I], according to
the equation K app
m ¼ K m (1 + [I]/K i ).
Trang 9Pyruvate 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 30C
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 25C
with the analogue 1c in the presence of Mn2+(reported
t1/2¼ 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
D I S C U S S I O N
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
analogues of PEP are those differing by substitutions at
C-3 (1b–e) Modifications of the phosphate and the
carboxylate groups resulted in inactive compounds, in
general terms These two anionic centres probably
contrib-ute to the chelation of the metal required for binding to
these enzymes; disruption of one contact may suffice to
abolish binding This is the most likely explanation of the
fact that, in a general sense, the best inhibitors among
compounds 2a–i were those preserving a negative charge in
the modified position, namely compounds 2b and 2f The
preference of these enzymes for the Z- (like 1c) over the
E-steroisomer (1d) is also a common feature Only enolase did not discern between the two isomers
According to the data presented in Table 2, enzyme Iof the PTS imposes the most stringent geometrical restrictions
to its substrate This protein is thought to be active only as a dimer, and its dimerization is induced by the PEP molecule [49] Therefore, this process might become an alternative checkpoint for substrate binding, and reduce the chances of finding good inhibitors for this enzyme In fact, it might be possible that some of the screened compounds bind to the dimer without displaying inhibition because they cannot compete with PEP during the dimerization step Note that the inhibition assays were carried out at low enzyme I concentrations, where protein association plays an import-ant role Further experiments will be carried out under conditions where enzyme Iis predominantly a dimer in order to clarify this possibility
Pyruvate kinase is an intensively studied enzyme Most of the data presented in this work for this enzyme corroborates previous results However, some observations are of special interest For instance, the remarkable difference observed between the Z- and E-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 lM Ki for (Z)-phosphoenolbutyrate (1e) vs 49.5 lM 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-tion by adopting an alternative orientainhibi-tion in the active site, similar to that shown in Fig 4 A second possibility is that the modified group is embedded into a hydrophobic pocket
Fig 4 Alternative binding mode of inhibitors to PEP carboxylase (A) Schematic representation of PEP and bicarbonate bound into the active site (B) Suggested alternative binding mode for compounds 2b (shown), 2h and 2i.
Fig 3 Irreversible inhibition of enzyme I Inactivation 30 C induced
by 0.5 m M (Z)-3-Cl-PEP (h), 0.5 m M (E)-3-Cl-PEP (s), 5 m M 2g (n),
5 m M 3a ()), 5 m M 3c (I) and no inhibitor (j) The incubation
mix-ture contained 5 m M MgCl 2 , 1 m M D -glucose and catalytic amounts of
the rest of components of the PTS necessary to maintain multiple
turnovers.
Trang 10that 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
Mg2+ 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] I n that study, the
metal-free form of fosfomycin was proposed to compete
with free PEP for the enzyme’s allosteric site Similarly,
complex formation with Mg2+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
A C K N O W L E D G E M E N T S
We are indebted to the Swiss National Science Foundation (grant
31–45838.95) and the Secretarı´a de Estado de Educacio´n y
Universi-dades (Spain) for financial support Special thanks to Prof Ronald L.
Somerville (Purdue University) for his kind donation of (Z)-3-F-PEP
and (Z)-3-Me-PEP We also thank Dr Eloy Arenas Bernal for assisting
us with the HPLC work, and Prof Peter Bigler (University of Bern) for
his helpful advice with NMR experiments.
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