Báo cáo Y học: Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes pot

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 as reversible and irreversible inhibitors of Mg 2+ -andMn 2+ - activated PEP-utilizing enzymes: enzyme I of 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 I was 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 Mg 2+ , but not with Mn 2+ . 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 (t 1/2 ¼ 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 (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 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 I of 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 I of 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) 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- 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, methyl D , 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 TCI America. 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 (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- 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 pH ¼ 12.0 (step 3). Published methods were also followed for the synthesis of sulfoenolpyruvate (3a)[18],and a-(dihydroxyphosphonylmethyl) acrylic acid (3b)[6]. 1 H- and 13 C-NMR spectra were recorded at 300.1 and 75.47 MHz, respectively. Spectra in D 2 O were calibrated against sodium 3-(trimethyl)propane-1-sulfonate (external standard). 31 P-NMR at 81 MHz were calibrated against a 85% phosphoric acid external standard (d ¼ 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 H 2 Oat pH ¼ 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 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 1b 7.62 (dd, 73.5, 2.9) – 1c 6.72 (d, 1.1) – 1d 6.17 (d, 1.1) – 1e 6.32 (dq, 7.4, 2.2) 1.75 (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) 2e 5.16–5.12 (m, 2H) – 2f 5.23–5.08 (m, 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) 2i b 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. b 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), 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,R 2 ¼ Br, X ¼ Br), 1 h reaction at 60 °C, 2.54 g, 100% [22]. 6e: 2.0 g, 100% [23]. 7a:reactionat70°C for 1 h. Purified by FC (hexanes/ ethyl acetate, 1 : 1, v/v): 1.5 g, 58% yield, 4 : 1 mixture of the Z-andE-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. 1 H-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 0–4 °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- 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 (4 °C, 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 2h.The solution was kept at pH 12.5 for 5 h and then neutralized with 1 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- nium bicarbonate (0.2 M to 1 M in 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: ditriethylammonium salt, 0.11 g, 10%. Chloromethylphosphonate 3c Trimethylsilyl 2-trimethylsilyloxypropenoate (9)waspre- pared as previously described [22]. Chloromethylphosphon- ic acid dichloride (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 50 °C. The reaction mixture was refluxed for 4 h. Benzene was removed under vacuum, and the unstable cyclic acylphosphate 10 was Kugelrohr distilled at around 130 °C (0.1 mbar): 0.4 g, 22%, 1 H-NMR (CDCl 3 ), d:5.82 3228 L. F. Garcı ´ a-Alles and B. Erni (Eur. J. Biochem. 269) Ó FEBS 2002 (1H, dd, J ¼ 3.7, 1.8 Hz, CH 2 ¼ C), 5.54 (1H, d, 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 Unless otherwise indicated, all experiments were performed at 30 °C, in 96-well microtitre plates. Progress curves were recorded and the initial rates were calculated as the maximal slope of the absorption curve obtained. IC 50 values were measured using 0.1 m M PEP (0.1 m MD -2-phosphoglyceric acid in the case of enolase) and in the presence of 0–5 m M inhibitor at the enzyme and metal concentrations indicated below. Enzyme I activity 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 l M enzyme I, 1 l M HPr, 20 l M IIA Glc ,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 -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 m M MgCl 2 or 0.05 UÆmL )1 of pyruvate kinase and 1 m M MnCl 2 . 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 )1 of PEP carboxylase and either 5 m M MgCl 2 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 enolase and 2 m M MnCl 2 . Enzyme inactivation experiments The time-dependent inactivation assays were carried out under turnover conditions. The enzymes (5 l M enzyme I, 3UÆmL )1 pyruvate kinase, 1.1 UÆmL )1 PEP carboxylase or 2UÆmL )1 enolase) were preincubated for 10 min at 30 °C in the presence of enough of the rest of components to maintain multiple turnovers (as indicated above). MgCl 2 (5 m M ) was present during the incubation (also 0.5 m M MnCl 2 with PEP carboxylase and enolase) together with 0.5 m M 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 1-chloromethyl-vinyl derivative 6e, by nucleophilic displace- ment with potassium thioacetate. Subsequent replacement 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 (R 2 ¼ CH 2 CO 2 H, X ¼ Cl) and 4g (R 2 ¼ CH 2 OH, X ¼ 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 2hat37°C in water-saturated t BuOMe (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 LypozymeÒ. 1-Chloro-3-hydroxy-propan- 2-one (4g)wasobtainedin74%yieldafterFCwith 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 [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,and2g 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.forthe preparation of pure (Z)-3-chlorophosphoenolpyruvate (1c) from 3,3-dichloropyruvic acid (5b,R 2 ¼ CO 2 H, X ¼ 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-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 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 ,20m M KH 2 PO 4 ,pH¼ 6.0, KCl (0 m M for 2minto360m 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) 13 C-NMR spectra in CD 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 characterized by 1 H- and 31 P-NMR, in spite of its instability. As expected, hydrolysis of 10 produced compound 3c. Characterization of PEP analogues Compounds 1–3 were characterized by 1 H (see Table 1), 13 C- and 31 P-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.tobetheZ-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 ( 3 J HC )between the carboxylic carbon atom and the vinyl proton (HOO 13 C- C ¼ C- 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 J HC ¼ 7.2 Hz, 3 J PC ¼ 5.7 Hz) was assigned as the E-isomer and the compound with the weakest coupling ( 3 J HC ¼ 1.6 Hz, 3 J PC ¼ 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., D 2 O, pH ¼ 7.0) upfield from the vinyl signal of the Z-isomer (6.72 p.p.m., D 2 O, pH ¼ 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 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 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 I in 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)and3-Cl-PEP(1c)weakly inhibit enzyme I. Inhibition by these compounds is com- petitive and the same K i ¼ 0.4 m M was calculated for the two compounds [Fig. 2A, only shown for (Z)-3-F-PEP]. Inhibition by analogues with a hydroxymethylene (2d)ora phosphonate group (2f) instead of the carboxylate of PEP, and by D , 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 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 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 Ó FEBS 2002 Inhibitors of phosphoenolpyruvate-utilizing enzymes (Eur. J. Biochem. 269) 3231 to 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 Mg 2+ -activated pyruvate kinase [25]. We have found, however, that this compound becomes a strong inhibitor when Mn 2+ is present. Under such conditions, 2f inhibits competitively pyruvate kinase with a K i ¼ 80 l M (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 C 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 for 1e [43]. The results also highlight a common feature of PEP-utilizing enzymes: the preference for Z-over E-isomers (compare IC 50 values measured for 1c and 1d). In presence of Mg 2+ 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 K i of 2.2 m M [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 Mn 2+ . The inhibition constant for the 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 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 Enzyme I PKase PEPCase Enolase IC 50 IC 50 IC 50 IC 50 b Mg 2+ Mn 2+ t 50 Mg 2+ Mn 2+ t 50 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 >10 >10 >10 2b >10 >10 >10 >10 >10 0.9 1.2  8 2c >10 >10 >10 >10 >10 >10 >10 >10 2d >10  6 >10 >10 35% c 2 >10 >10 2e >10 >10 >10 >10 55% c >10 >10 >10 2f  8>10  9 0.03 5 3 1.4 0.3 2g >10 >10 ND >10 >10 ND 60% c 28% c 240 d >10 >10 ND 2h >10 >10 >10 >10 >10 0.8 >10 >10 2i >10 >10 >10  9 >10 1.2 >10 >10 Modified in the phosphate position 3a >10 >10 ND 5 1.2 >10 >10 ND >10 >10 3b >10 >10  6 >10 0.2 0.1 0.3 9 · 10 )3 3c >10 >10 ND >10 >10 ND  9 3 ND >10 >10 ND a Incubation also in the presence of 0.5 m M MnCl 2 . 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+ was replaced by Mn 2+ [18]. Five compounds, among those modified at the carboxylic position, inhibit moderately Mn 2+ -activated PEP carboxylase. In presence of this metal, inhibition by compounds 2b (not shown), 2h (not shown) and 3c (Fig. 2C) are competitive with K i values of 1.1, 1.2 and 3.9 m M , 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 Mn 2+ . 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)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 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 K i of 2.2 m M [25]. The same type of inhibition and similar inhibition constant value was measured for 2b in the presence of both Mg 2+ (K i ¼ 2.0 m M , not shown) and Mn 2+ (K i ¼ 2.2 m M , 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 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 (t 1/2 ) 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 (t 1/2 ¼ 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 I by 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 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 (triangles) and 3 m M (stars) analog 2b. PEP concentrations ( D -2-phosphoglyceric acid with enolase) were varied between 0 and 2m 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 ). 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 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)overthe 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 I of 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 I is 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-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- tion by adopting an alternative orientation 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), 5m M 3a ()), 5 m M 3c (I) and no inhibitor (j). The incubation mix- ture contained 5 m M MgCl 2 ,1m MD -glucose and catalytic amounts of the rest of components of the PTS necessary to maintain multiple turnovers. 3234 L. F. Garcı ´ 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 ´ a de Estado de Educacio ´ nyUniversi- 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. 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Synthesis of phospho enol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes Luis Fernando Garcı ´ a-Alles and Bernhard. UÆmL )1 of pyruvate kinase and 5 m M MgCl 2 or 0.05 UÆmL )1 of pyruvate kinase and 1 m M MnCl 2 . PEP carboxylase activity was determined in a coupled assay