ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 280, No. 1, July, pp. 40-44,199O
Synthesis of Oxalyl Phosphate and Processing of the Acyl Phosphate by PhosphoenolpyruvateDependent Enzymes’ James L. Kofron and George H. Reed2 The Institute for Enzyme Research, Graduate School, and Department of Biochemistry, and Life Sciences, The University of Wisconsin-Madison, Madison, Wisconsin 53705
School of Agricultural
Received January 8,199O
The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4°C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate. o 1990 Academic Press, Inc.
P-enolpyruvate3 possesses the highest phospho group transfer potential among the phosphorylated metabolites, and there has been continuing interest in mechai This work was supported, in part, by a grant from the National Institutes of Health, GM 35752. ’ To whom correspondence should be addressed at The Institute for Enzyme Research, 1710 University Avenue, Madison, WI 53705. s Abbreviations used: P-enolpyruvate, phosphoenolpyruvate; E,, the phosphorylated form of pyruvate, phosphate dikinase; ATPBS, adenosine-5’-0-(2.thiotriphosphate); ADPBS, adenosine-5’.0-(2.thiodiphosphate). 40
nistic aspects of the enzyme-mediated reactions of this compound. Various analogues of P-enolpyruvate have provided insight into the stereoselectivities of the active sites of enzymes which process this compound and into the chemical mechanisms of the enzymatic processes. These analogues may be grouped into three main classes: (i) compounds in which the C3 position has been modified (l-4), (ii) compounds in which the phosphate group has been altered (5-8), and (iii) compounds in which the CZ is an .sp3center (9-11). Most of these analogues serve as slow substrates or inhibitors for enzymes that process P-enolpyruvate. The weak substrate activities of the various phosphoenol derivatives is not well understood, although steric factors appear to be a logical source of interference. 1-Carboxyallenyl phosphate, an exception to the low activity trend, exhibits a respectable k,, , relative to P-enolpyruvate, with pyruvate kinase (4). Oxalate is a potent inhibitor of several enzymes that generate enolpyruvate as a transient product (12). The high affinity of these enzymes for oxalate is attributed to similarities between oxalate and the enolate dianion of pyruvate. The mixed anhydride of oxalic and phosphoric acid, oxalyl phosphate, would be virtually isosteric with P-enolpyruvate (see Scheme 1). Furthermore, the anticipated high phospho group transfer potential of this acyl phosphate offers the possibility for thermodynamically favorable reactions of this analogue in enzyme-catalyzed phospho transfers. There is prior evidence for a pyruvate kinase-dependent phosphorylation of oxalate by ATP in the observation that oxalate enhances an enzyme-dependent rate of positional isotope exchange in the &y-bridging and ,&nonbridging oxygens of ATP (13). The present paper describes the synthesis and characterization of oxalyl phosphate as well as results from assays in which oxalyl phosphate was tested as a substrate with pyruvate kinase; pyruvate, phosphate dikinase; and P-enolpyruvate carboxykinase. 0003-9%x/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROPERTIES
OF OXALYL
41
PHOSPHATE
RESULTS
AND
DISCUSSION
Synthesis and characterization of oralyl phosphate. Oxalyl phosphate was synthesized by gradual addition of 1.0 ml of oxalyl chloride4 to 10 ml of a vigorously stirring solution of 2 M K,HPO, in an ice bath. The competing _(-yp-o . O~kO I I hydrolysis reaction of oxalyl chloride is known to pro-0 -0 duce CO, COZ, and HCl(l6). A vigorous evolution of gas SCHEME 1 occurred upon addition of oxalyl chloride to the aqueous solution of phosphate. The presence of CO and CO2 in the gas was confirmed by gas phase FTIR spectroscopy. Yields of oxalyl phosphate, based on oxalyl chloride, EXPERIMENTAL PROCEDURES were lo-15%. The pH of the solution was between 3 and Materials. Pyruvate kinase was isolated from rabbit skeletal mus5 after addition of oxalyl chloride. The solution was cle by the method of Tietz and Ochoa (14). The enzyme was further stirred until it had cooled to 0°C (-10 min), by which purified by gel filtration chromatography (Sephacryl S-200) as detime a white precipitate of KH2P04 had formed. The rescribed previously (10). Enzyme which was used in the assays had a sulting suspension was centrifuged to remove the precipspecific activity of 200 IU rng-’ when measured in the coupled assay itate; the supernatant was diluted 50-fold with ice-cold with lactate dehydrogenase at 25”C, pH 7.5. Pyruvate, phosphate dikinase was isolated from Clostridium symbiosum as described previously distilled water and applied to a 60-ml column of Bio-Rad (15). The enzyme had specific activities of 12-24 IU rng-’ when asAGl-X8 200-400 mesh (Cll form) at 4°C. A linear gradisayed in the direction of ATP formation in a coupled assay with lactate ent of KC1 (100-800 mM, 0.5 1 total) was used to elute dehydrogenase. P-enolpyruvate carboxykinase from chicken liver was the compound from the column. Oxalyl phosphate was a gift from Dr. Thomas Nowak, University of Notre Dame. The endetected in the eluent fractions by 31P NMR. Fractions zyme had a specific activity of 6 IU rng-’ when measured in the direction of GTP formation, using a coupled assay with malate dehydrogecontaining oxalyl phosphate that were not used immedinase. Glucose-6-phosphate dehydrogenase, hexokinase, and oxalate ately were kept at 4°C. Attempts to obtain a solid salt decarboxylase were from Sigma. Lactate dehydrogenase and malate of oxalyl phosphate by evaporation of solutions under dehydrogenase were from Boehringer-Mannheim. Oxalyl chloride was reduced pressure, precipitation with organic solvents, or from Aldrich Chemical Company. lyophilization were without success. Acetyl phosphate is Assays. The viability of oxalyl phosphate as a substrate for pyruknown to convert to PPi and acetate at high ionic vate kinase was tested in a coupled assay with hexokinase and glucosestrength (17, 18), and all of the above methods produce 6-phosphate dehydrogenase. Fixed point assays were used for measurement of the extent of reaction in the opposite direction. Oxalate high concentrations of salt at some point in the proce(10 mM) and ATP (3.0 mM) were incubated with pyruvate kinase (0.04 dure. At room temperature (-23°C) and pH 5.0, the to 46 mg ml-‘) in a buffer consisting of 50 mM Hepes/KOH, pH 7.5, half-life of oxalyl phosphate is 2.5 h. 100 mM KC1 (Buffer A), 5 mM MgCl,, and 1 mM MnCl,. Aliquots were Oxalyl phosphate was characterized by ‘IP NMR (6 removed at fixed intervals, the protein was precipitated by addition of = -0.5 ppm at pH 8) and 13C NMR (6 = 161.2 ppm, JCp chloroform, and the ADP/ATP ratios in the supernatant were determined by ion exchange HPLC (Whatman Partisil-10 SAX column). = 10 Hz; 6 = 163.7 ppm, Jc, = 8 Hz at pH 4.5). The pair A similar assay was used to determine the extent of the enzyme-cataof equally intense 13Cdoublets shows that the product is lyzed reaction of Rr ATPpS with oxalate except that CdCl, replaced an acyclic, monophosphorylated derivative (see Scheme MnCl, in the assay mixtures. I). Solutions gave a positive reaction with hydroxylFormation of GTP due to substrate activity of oxalyl phosphate amine and FeC& in the calorimetric assay for acyl phoswith P-enolpyruvate carboxykinase was assayed by ion exchange phates (19). A pH titration of oxalyl phosphate between HPLC. The assay mixture contained 50 mM Tris/HCl, pH 7.4, 100 mM KCI, 2 mM MnCl,, 1 mM GDP, and 200 mM KHCO,. The reaction pH 12 and 1.5 shows a single protonation with a pK, of was initiated by the addition of P-enolpyruvate carboxykinase (80 Fug 4.5. The 31P NMR chemical shift of the oxalyl phosphate ml. ‘) and oxalyl phosphate (10 mM). Aliquots of the reaction mixture changes by -5 ppm as a result of this protonation. were stopped at various time points by chloroform-induced precipitation of the protein. The content of GTP in the supernatant was quanHydrolysis of oxalylphosphate. Rates of hydrolysis of titated by ion exchange HPLC. oxalyl phosphate were studied over a range of pH values Spectroscopic measurements. EPR spectra were obtained at 35 at 35°C (see Fig. 1). The observed first-order rate conGHz with a Varian E-109-Q spectrometer. i3C NMR spectra were obstant for the hydrolysis of oxalyl phosphate at pH 7.0 tained at natural abundance with a Bruker AM-400. 31P NMR spectra and 35°C is 2.0 X lop2 min..‘. The corresponding values were obtained at 24.3 MHz and at 162 MHz with a Varian NV-14 and for formyl phosphate and acetyl phosphate are 1.1 X lop3 a Bruker AM-400, respectively. 13Cchemical shifts were measured relmini’ (20) and 1.8 X lop2 min-’ (17, 21), respectively. ative to an external standard of dioxane and referenced to tetramethylsilane. 31P chemical shifts are reported relative to an external stanThe pH range over which the rate of hydrolysis of oxalyl
OAO I
dard of 85% phosphoric acid. Concentrations of oxalyl phosphate were determined by 31P NMR using an external standard of PI’,. The positions of bond cleavage upon hydrolysis of oxalyl phosphate in Hi”0 were determined by measurement of the 1RO:‘60 ratio in the product, P I> bv_ =P NMR.
4 All manipulations a fume hood. Oxalyl
(16).
that involved oxalyl chloride were performed in chloride releases CO upon reaction with water
42
KOFRON
AND
REED
@ 54-
?r( /= i” LL.
32-
-100 14 0
”
I 2
”
I,, 4
l...I...I.~~I~~ll 6 8
10
12
14
PH FIG. 1. The pH dependence of the first order rate constant for hydrolysis of oxalyl phosphate at 35°C. The concentrations of oxalyl phosphate were measured spectrophotometrically following conversion to the Fe(II1) complex of the hydroxamate (18). The solid curve is drawn through the experimental data points.
Id 0
100 l/lOxaIyl
200
300
Phosphate]. L/mmol
FIG. 2. Initial velocity data for pyruvate kinase-catalyzed reaction of oxalyl phosphate with ADP at 22°C. The assay mixture included 50 mM Hepes/KOH, pH 7.5, 100 mM KCl, 20 IU glucose-6-phosphate dehydrogenase, -50 IU hexokinase, 5 mM ADP, 0.2 mM NAD+, 5 mM glucose, 15 mM MgClz, 5 mM MnCl,, and 15 pg ml-’ pyruvate kinase.
tive transition states. Between pH 12 and 13 the hydrolysis rate of oxalyl phosphate increases (see Fig. 1). The >95% C-O bond cleavage observed at pH 12.5 indicates phosphate is at a minimum is wider than that for formyl that hydrolysis proceeds through an associative nucleophosphate; the rate of hydrolysis of formyl phosphate increases sharply below pH 2.5 and above pH 10 (21). philic attack by OH- at the carbonyl center. The extra negative charge carried by oxalyl phosphate Substrate properties of oxalyl phosphate with pyruvate probably accounts for the increased stability over formyl kinase. Results from a coupled assay (hexokinase/gluphosphate in basic solutions. case-6-phosphate dehydrogenase) show that oxalyl The temperature profiles of the rates of hydrolysis of phosphate supports the pyruvate kinase-dependent prooxalyl phosphate were measured at pH 1.5,5.0, and 12.1 duction of ATP from ADP. A dependence of ATP proin order to determine activation energies for the hydroduction on the presence of oxalyl phosphate eliminates possible trace contaminating adenylate kinase as the lysis reactions. Activation parameters for the hydrolysis of oxalyl phosphate are collected in Table I. The value source of the activity. The rate varies linearly with the concentration of pyruvate kinase. Progress curves were of AS+ (-0 e. u.) at pH 5.0 and the predominance of Plinear for -1 min, after which product inhibition beO bond cleavage (X30%) suggest that, at this pH, the came apparent. Kinetic constants, K, = 11 f 2 PM and transition state for the hydrolysis has significant dissok cat = 2 min-l, were obtained from a double reciprocal ciative character (22). At pH 1.5, the value of -21 e. u. plot of the initial velocity data (see Fig. 2). Given the for ASS, coupled with a large percentage of C-O bond potent inhibitory properties of the hydrolysis product of cleavage in Hi*0 (>70%) indicates that the predominant oxalyl phosphate, the initial velocities might be subject mechanism of hydrolysis changes to that of an associato inhibition from an oxalate contamination of the subtive nucleophilic attack of water at the carbonyl carbon. strate. 13CNMR, however, showed that the extent of hyThe entropy of activation obtained at pH 12.1 (ASS drolysis of freshly prepared solutions of oxalyl phos= -7 e. u.) is intermediate for associative and dissociaphate that were kept on ice for -30 min was t5%. The initial velocities obtained in assays with fresh preparations of oxalyl phosphate were virtually identical to TABLE I those obtained with oxalyl phosphate from the Dowex Activation Parameters for Hydrolysis of Oxalyl Phosphate” chromatographic treatment. Furthermore, solutions of oxalyl phosphate that had been incubated with oxalate % c-o decarboxylase (40 IU ml-‘) immediately before use gave AH* AS+ bond AG* (kcal/mol) cleavage * initial velocities that were likewise identical to those obat 23°C (kcal/mole) (e.u.) PH tained in the previous experiments. The apparent K,,, for oxalyl phosphate is comparable to the Ki (6 PM) for oxa1.5 22.2 f 1.6 16.0 + 0.9 -21 f 3 >70 5.0 23.2 f 1.0 23.2 + 1.0 0+2 95 present as a trace contaminant in the oxalyl phosphate, is primarily responsible for the low initial velocities. a Error limits represent standard deviations. The reverse reaction (i.e., phosphorylation of oxalate * The percentage of C-O bond cleavage was determined by 31P NMR by ATP) is sufficiently unfavorable, thermodynamically, analysis of Pi formed from hydrolysis of oxalyl phosphate in H,‘sO.
PROPERTIES
OF OXALYL
to complicate characterization. Even in samples that have been treated to minimize bicarbonate content, the inherent bicarbonate-dependent ATPase (14) activity of pyruvate kinase typically produces higher levels of ADP than the equilibrium for the reaction of ATP and oxalate. Moreover, the low equilibrium level of oxalyl phosphate is difficult to detect in the hydroxamate assay. Oxalate and bicarbonate compete for the same binding site on the enzyme (23) such that oxalate effectively inhibits the ATPase activity. Measurements (see Experimental Procedures) of ADP/ATP ratios in assay mixtures of ATP, oxalate, metal cofactors, and enzyme (0.04 to 46 mg ml-‘), which were allowed to equilibrate at room temperature for up to 2 h, showed that the ADP/ATP ratio was co.02 * 0.01. This low apparent extent of reaction could stem from either kinetic or thermodynamic limitations. Significantly longer incubation times were, however, impractical due to the relatively short half-life of oxalyl phosphate under these conditions. Enzyme-catalyzed phosphorylation of oxalate was readily demonstrated in experiments with the P-thio analogue of ATP, ATPPS. Lerman and Cohn (24) demonstrated that the higher y-phospho group transfer potential of ATPPS, relative to ATP, can be used to characterize enzymatic reactions for which equilibrium lies far in the direction of ATP. Assays for ADP/3S/ ATPpS in reaction mixtures of oxalate (10 mM), MgCl, (4 mM), CdClz (1 mM), enzyme (46 mg ml-‘), and Rp ATPpS (3 mM) for 2.5 h, provided an estimate” of an equilibrium constant:
[ATPPSI [OxI [ox _ pl
Ic:!jz, = [ADPpSl
= 2.0 f 0.5 x 102.
Nucleoside phosphorothioates typically react more slowly than their oxynucleotide counterparts (25). The readily measurable extent of reaction of ATPpS and oxalate, under assay conditions similar to those used for the analogous reaction with ATP, suggests that a thermodynamic, rather than a kinetic, factor limits the extent of reaction of ATP and oxalate. The low extent of reaction of ATP and oxalate is consistent with the expected 60-fold ratio of equilibrium constants for reactions of ATP and ATP@ (24). The conditional equilibrium constant, K’$$, for the reaction of ADP@ with Penolpyruvate corrected to pH 7.5 is -170 (24). These results suggest that the phospho group transfer potential of oxalyl phosphate is comparable to that of P-enolpyruvate at pH 7.5. ’ The calculation assumes that the reaction is at equilibrium. Due to the high concentration of enzyme (0.77 mM) relative to the concentrations of substrates, the position of equilibrium may be influenced by differential binding affinities of substrates and products to the enzyme.
43
PHOSPHATE
A
I I 200 Gauss FIG. 3. EPR spectra at 35 GHz for Mn(I1) in solutions of pyruvate, phosphate dikinase, and (A) P-enolpyruvate and oxalate or (B) oxalyl phosphate. Samples contained 20 mM Hepes/NH,OH, pH 6.8,75 mM NH&l, 175 mg ml-’ enzyme, and 0.44 mM MnClx. Sample (A) was and 4.3 mM oxalate generated by addition of 1.7 mM P-&olpyruvate to form the Es-Mn (II)-oxalate complex. Sample (B) was generated by addition of 4 mM oxalyl phosphate to the solution of enzyme and MnC&. The ionic composition of the solution for (B) includes KC1 (-20 mM) that was present in the stock solution of oxalyl phosphate. Spectra were obtained at 2°C.
Substrate properties of oxalylphosphate withpyruuate, phosphate dikinase. The characteristic EPR spectrum for the E,-Mn(II)-oxalate complex (15) provided a sensitive means for detection of phosphorylation of the active site histidyl residue by oxalyl phosphate. Contamination of the oxalyl phosphate by oxalate and phosphate was not a concern in this assay because EPR spectra of the enzyme-Mn(II)-oxalate and enzyme-Mn(II)-Pi species do not resemble the spectrum of the E,-Mn(II)oxalate complex (15). The appearance of this distinctive spectrum for the sample of oxalyl phosphate, enzyme, and Mn(I1) (Fig. 3) indicates that oxalyl phosphate reacts to give oxalate bound to the E, form of the enzyme. Multiple turnovers were not observed with oxalyl phosphate in the direction of formation of Pi and ATP probably due to the unusual stability of the E,-Mn(II)oxalate complex (26). A single-turnover experiment was performed in order to confirm that oxalyl phosphate donated its phospho group to the histidyl residue on the enzyme. A solution containing enzyme and MnCl, was
44
KOFRON AND REED
incubated with oxalyl phosphate. An excess of EDTA was added to sequester Mn(I1) and thereby facilitate dissociation of oxalate from the complex. The resulting mixture was chromatographed over a column of Sephadex G-25 to separate the protein from EDTA and oxalate. Fractions containing protein were pooled and incubated with MnClz, AMP, and PPi. Protein was precipitated by addition of chloroform, and ATP in the supernatant was quantitated by HPLC. Approximately one equivalent (0.95) of ATP was formed per equivalent of dimeric enzyme. Substrate properties of oxalyl phosphate with phosphoenolpyruvate carboxykinase. The activity of oxalyl phosphate with P-enolpyruvate carboxykinase was investigated by a fixed-point assay for formation of GTP. The sample containing oxalyl phosphate and enzyme converted about 40% of the GDP to GTP in 10 min. There was no detectable GTP in experiments with control samples that lacked either enzyme or oxalyl phosphate. Conclusions. Solutions of oxalyl phosphate can be prepared in satisfactory yields by reaction of oxalyl chloride with Pi in aqueous medium. The stability of the compound at neutral pH is sufficient to allow spectroscopic and enzymatic characterization. The acyl phosphate has a high phospho group transfer potential and serves as a phospho group donor in reactions catalyzed by pyruvate kinase, pyruvate, orthophosphate dikinase, and P-enolpyruvate carboxykinase. With pyruvate kinase, however, kcat for oxalyl phosphate reaction with ADP is ~0.02% of that for the corresponding reaction with P-enolpyruvate and ADP. The k,,, for phosphorylation of ADP is also only 1% of the k,,, reported for the oxalate-stimulated positional isotope exchange in ATP (13). Although nonproductive binding can be invoked to account for the low kcat, it is possible that kcat for oxalyl phosphate is limited by release of products. The product complex, enzyme-oxalate-Mn( II)-ATP-Mg( II), is known to have a high affinity for the enzyme (27). The value of kcat for oxalyl phosphate with pyruvate kinase falls into the lower end of the range of constants reported for other analogues of P-enolpyruvate (l-4). ACKNOWLEDGMENTS The authors thank Jenny L. Buchbinder for providing samples of nucleoside phosphorothioates and Dr. Thomas Nowak for the sample of P-enolpyruvate carboxykinase. High-field NMR spectra were ob-
tained at the National Grant RR02301).
Magnetic
Resonance Facility
at Madison
(NIH
REFERENCES 1. Stubbe, J., and Kenyon, G. L. (1972) Biochemistry 11,338-345. 2. Stubbe, J., and Kenyon, G. L. (1971) Biochemistry 10,2669-2677. 3. Wirsching, P., and O’Leary, M. H. (1988) Biochemistry 27,13481355. 4. Wirsching, P., and O’Leary, M. H. (1988) Biochemistry 27,13551360. 5. Lazarus, R. A., Benkovic, P. A., and Benkovic, S. J. (1979) Arch. Biochem. Biophys. 197,218-225. 6. Sikkema, K. D., and O’Leary, M. H. (1988) Biochemistry 27, 1342-1347. 7. Hansen, D. E., and Knowles, J. R. (1982) J. Biol. Chem. 257, 14,795-14,799. 8. Peliska, J. A., and O’Leary, M. H. (1989) Biochemistry 28,16041611. 9. Nowak, T., and Mildvan, A. S. (1970) Biochemistry 16, 1343-
1350. 10. Ash, D. E., Goodhart, P. J., and Reed, G. H. (1984) Arch. Biochem. Biophys. 228,31-40. 11. Kayne, F. J. (1974) Biochem. Biophys. Res. Commun. 59,8-13. 12. Reed, G. H., and Morgan, S. D. (1974) Biochemistry 13, 35373541. 13. Lowe, G., and Sproat, B. S. (1979) J. Chem. Sot., Perkin Trans. 1, 1622-1630. 14. Tietz, A., and Ochoa, S. (1958) Arch. Biochem. Biophys. 78,477493. 15. Kofron, J. L., Ash, D. E., and Reed, G. H. (1988) Biochemistry 27, 4781-4787. 16. Staudinger, H., (1908) Ber. 41,355s. 17. DiSabato, G., and Jencks, W. P. (1961) J. Amer. Chem. Sot. 83, 4400-4405. 18. Herschlag, D., and Jencks, W. P. (1986) J. Amer. Chem. Sot. 108,
7938-7946. (Colowick, 19. Stadtman, E. R. (1957) in Methods in Enzymology S. P., and Kaplan, N. O., Eds.), Vol. 3, pp. 228-231, Academic Press, San Diego. 20. Jahansouz, H., Mertes, K. B., Mertes, M. P., and Himes, R. H. (1989) Bioorg. Chem. 17,207-216. 21. Lynen, F. (1940) Ber. 73,367-372. 22. Herschlag, D., and Jencks, W. P. (1986) J. Amer. Chem. Sot. 109, 4665-4674. 23. Lodato, D. T. (1986) Ph.D. Thesis, University of Pennsylvania. 24. Lerman, C. L., and Cohn, M. (1980) J. Biol. Chem. 255, 87568760. 25. Cohn, M. (1982) Act. Chem. Res. 15,326-332. 26. Michaels, G., Milner, Y., and Reed, G. H. (1976) Biochemistry 14, 321223219. 26, 224327. Lodato, D. T., and Reed, G. H. (1987) Biochemistry 2250.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 280, No. 1, July, pp. 40-44,199O
Synthesis of Oxalyl Phosphate and Processing of the Acyl Phosphate by PhosphoenolpyruvateDependent Enzymes’ James L. Kofron and George H. Reed2 The Institute for Enzyme Research, Graduate School, and Department of Biochemistry, and Life Sciences, The University of Wisconsin-Madison, Madison, Wisconsin 53705
School of Agricultural
Received January 8,199O
The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4°C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate. o 1990 Academic Press, Inc.
P-enolpyruvate3 possesses the highest phospho group transfer potential among the phosphorylated metabolites, and there has been continuing interest in mechai This work was supported, in part, by a grant from the National Institutes of Health, GM 35752. ’ To whom correspondence should be addressed at The Institute for Enzyme Research, 1710 University Avenue, Madison, WI 53705. s Abbreviations used: P-enolpyruvate, phosphoenolpyruvate; E,, the phosphorylated form of pyruvate, phosphate dikinase; ATPBS, adenosine-5’-0-(2.thiotriphosphate); ADPBS, adenosine-5’.0-(2.thiodiphosphate). 40
nistic aspects of the enzyme-mediated reactions of this compound. Various analogues of P-enolpyruvate have provided insight into the stereoselectivities of the active sites of enzymes which process this compound and into the chemical mechanisms of the enzymatic processes. These analogues may be grouped into three main classes: (i) compounds in which the C3 position has been modified (l-4), (ii) compounds in which the phosphate group has been altered (5-8), and (iii) compounds in which the CZ is an .sp3center (9-11). Most of these analogues serve as slow substrates or inhibitors for enzymes that process P-enolpyruvate. The weak substrate activities of the various phosphoenol derivatives is not well understood, although steric factors appear to be a logical source of interference. 1-Carboxyallenyl phosphate, an exception to the low activity trend, exhibits a respectable k,, , relative to P-enolpyruvate, with pyruvate kinase (4). Oxalate is a potent inhibitor of several enzymes that generate enolpyruvate as a transient product (12). The high affinity of these enzymes for oxalate is attributed to similarities between oxalate and the enolate dianion of pyruvate. The mixed anhydride of oxalic and phosphoric acid, oxalyl phosphate, would be virtually isosteric with P-enolpyruvate (see Scheme 1). Furthermore, the anticipated high phospho group transfer potential of this acyl phosphate offers the possibility for thermodynamically favorable reactions of this analogue in enzyme-catalyzed phospho transfers. There is prior evidence for a pyruvate kinase-dependent phosphorylation of oxalate by ATP in the observation that oxalate enhances an enzyme-dependent rate of positional isotope exchange in the &y-bridging and ,&nonbridging oxygens of ATP (13). The present paper describes the synthesis and characterization of oxalyl phosphate as well as results from assays in which oxalyl phosphate was tested as a substrate with pyruvate kinase; pyruvate, phosphate dikinase; and P-enolpyruvate carboxykinase. 0003-9%x/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROPERTIES
OF OXALYL
41
PHOSPHATE
RESULTS
AND
DISCUSSION
Synthesis and characterization of oralyl phosphate. Oxalyl phosphate was synthesized by gradual addition of 1.0 ml of oxalyl chloride4 to 10 ml of a vigorously stirring solution of 2 M K,HPO, in an ice bath. The competing _(-yp-o . O~kO I I hydrolysis reaction of oxalyl chloride is known to pro-0 -0 duce CO, COZ, and HCl(l6). A vigorous evolution of gas SCHEME 1 occurred upon addition of oxalyl chloride to the aqueous solution of phosphate. The presence of CO and CO2 in the gas was confirmed by gas phase FTIR spectroscopy. Yields of oxalyl phosphate, based on oxalyl chloride, EXPERIMENTAL PROCEDURES were lo-15%. The pH of the solution was between 3 and Materials. Pyruvate kinase was isolated from rabbit skeletal mus5 after addition of oxalyl chloride. The solution was cle by the method of Tietz and Ochoa (14). The enzyme was further stirred until it had cooled to 0°C (-10 min), by which purified by gel filtration chromatography (Sephacryl S-200) as detime a white precipitate of KH2P04 had formed. The rescribed previously (10). Enzyme which was used in the assays had a sulting suspension was centrifuged to remove the precipspecific activity of 200 IU rng-’ when measured in the coupled assay itate; the supernatant was diluted 50-fold with ice-cold with lactate dehydrogenase at 25”C, pH 7.5. Pyruvate, phosphate dikinase was isolated from Clostridium symbiosum as described previously distilled water and applied to a 60-ml column of Bio-Rad (15). The enzyme had specific activities of 12-24 IU rng-’ when asAGl-X8 200-400 mesh (Cll form) at 4°C. A linear gradisayed in the direction of ATP formation in a coupled assay with lactate ent of KC1 (100-800 mM, 0.5 1 total) was used to elute dehydrogenase. P-enolpyruvate carboxykinase from chicken liver was the compound from the column. Oxalyl phosphate was a gift from Dr. Thomas Nowak, University of Notre Dame. The endetected in the eluent fractions by 31P NMR. Fractions zyme had a specific activity of 6 IU rng-’ when measured in the direction of GTP formation, using a coupled assay with malate dehydrogecontaining oxalyl phosphate that were not used immedinase. Glucose-6-phosphate dehydrogenase, hexokinase, and oxalate ately were kept at 4°C. Attempts to obtain a solid salt decarboxylase were from Sigma. Lactate dehydrogenase and malate of oxalyl phosphate by evaporation of solutions under dehydrogenase were from Boehringer-Mannheim. Oxalyl chloride was reduced pressure, precipitation with organic solvents, or from Aldrich Chemical Company. lyophilization were without success. Acetyl phosphate is Assays. The viability of oxalyl phosphate as a substrate for pyruknown to convert to PPi and acetate at high ionic vate kinase was tested in a coupled assay with hexokinase and glucosestrength (17, 18), and all of the above methods produce 6-phosphate dehydrogenase. Fixed point assays were used for measurement of the extent of reaction in the opposite direction. Oxalate high concentrations of salt at some point in the proce(10 mM) and ATP (3.0 mM) were incubated with pyruvate kinase (0.04 dure. At room temperature (-23°C) and pH 5.0, the to 46 mg ml-‘) in a buffer consisting of 50 mM Hepes/KOH, pH 7.5, half-life of oxalyl phosphate is 2.5 h. 100 mM KC1 (Buffer A), 5 mM MgCl,, and 1 mM MnCl,. Aliquots were Oxalyl phosphate was characterized by ‘IP NMR (6 removed at fixed intervals, the protein was precipitated by addition of = -0.5 ppm at pH 8) and 13C NMR (6 = 161.2 ppm, JCp chloroform, and the ADP/ATP ratios in the supernatant were determined by ion exchange HPLC (Whatman Partisil-10 SAX column). = 10 Hz; 6 = 163.7 ppm, Jc, = 8 Hz at pH 4.5). The pair A similar assay was used to determine the extent of the enzyme-cataof equally intense 13Cdoublets shows that the product is lyzed reaction of Rr ATPpS with oxalate except that CdCl, replaced an acyclic, monophosphorylated derivative (see Scheme MnCl, in the assay mixtures. I). Solutions gave a positive reaction with hydroxylFormation of GTP due to substrate activity of oxalyl phosphate amine and FeC& in the calorimetric assay for acyl phoswith P-enolpyruvate carboxykinase was assayed by ion exchange phates (19). A pH titration of oxalyl phosphate between HPLC. The assay mixture contained 50 mM Tris/HCl, pH 7.4, 100 mM KCI, 2 mM MnCl,, 1 mM GDP, and 200 mM KHCO,. The reaction pH 12 and 1.5 shows a single protonation with a pK, of was initiated by the addition of P-enolpyruvate carboxykinase (80 Fug 4.5. The 31P NMR chemical shift of the oxalyl phosphate ml. ‘) and oxalyl phosphate (10 mM). Aliquots of the reaction mixture changes by -5 ppm as a result of this protonation. were stopped at various time points by chloroform-induced precipitation of the protein. The content of GTP in the supernatant was quanHydrolysis of oxalylphosphate. Rates of hydrolysis of titated by ion exchange HPLC. oxalyl phosphate were studied over a range of pH values Spectroscopic measurements. EPR spectra were obtained at 35 at 35°C (see Fig. 1). The observed first-order rate conGHz with a Varian E-109-Q spectrometer. i3C NMR spectra were obstant for the hydrolysis of oxalyl phosphate at pH 7.0 tained at natural abundance with a Bruker AM-400. 31P NMR spectra and 35°C is 2.0 X lop2 min..‘. The corresponding values were obtained at 24.3 MHz and at 162 MHz with a Varian NV-14 and for formyl phosphate and acetyl phosphate are 1.1 X lop3 a Bruker AM-400, respectively. 13Cchemical shifts were measured relmini’ (20) and 1.8 X lop2 min-’ (17, 21), respectively. ative to an external standard of dioxane and referenced to tetramethylsilane. 31P chemical shifts are reported relative to an external stanThe pH range over which the rate of hydrolysis of oxalyl
OAO I
dard of 85% phosphoric acid. Concentrations of oxalyl phosphate were determined by 31P NMR using an external standard of PI’,. The positions of bond cleavage upon hydrolysis of oxalyl phosphate in Hi”0 were determined by measurement of the 1RO:‘60 ratio in the product, P I> bv_ =P NMR.
4 All manipulations a fume hood. Oxalyl
(16).
that involved oxalyl chloride were performed in chloride releases CO upon reaction with water
42
KOFRON
AND
REED
@ 54-
?r( /= i” LL.
32-
-100 14 0
”
I 2
”
I,, 4
l...I...I.~~I~~ll 6 8
10
12
14
PH FIG. 1. The pH dependence of the first order rate constant for hydrolysis of oxalyl phosphate at 35°C. The concentrations of oxalyl phosphate were measured spectrophotometrically following conversion to the Fe(II1) complex of the hydroxamate (18). The solid curve is drawn through the experimental data points.
Id 0
100 l/lOxaIyl
200
300
Phosphate]. L/mmol
FIG. 2. Initial velocity data for pyruvate kinase-catalyzed reaction of oxalyl phosphate with ADP at 22°C. The assay mixture included 50 mM Hepes/KOH, pH 7.5, 100 mM KCl, 20 IU glucose-6-phosphate dehydrogenase, -50 IU hexokinase, 5 mM ADP, 0.2 mM NAD+, 5 mM glucose, 15 mM MgClz, 5 mM MnCl,, and 15 pg ml-’ pyruvate kinase.
tive transition states. Between pH 12 and 13 the hydrolysis rate of oxalyl phosphate increases (see Fig. 1). The >95% C-O bond cleavage observed at pH 12.5 indicates phosphate is at a minimum is wider than that for formyl that hydrolysis proceeds through an associative nucleophosphate; the rate of hydrolysis of formyl phosphate increases sharply below pH 2.5 and above pH 10 (21). philic attack by OH- at the carbonyl center. The extra negative charge carried by oxalyl phosphate Substrate properties of oxalyl phosphate with pyruvate probably accounts for the increased stability over formyl kinase. Results from a coupled assay (hexokinase/gluphosphate in basic solutions. case-6-phosphate dehydrogenase) show that oxalyl The temperature profiles of the rates of hydrolysis of phosphate supports the pyruvate kinase-dependent prooxalyl phosphate were measured at pH 1.5,5.0, and 12.1 duction of ATP from ADP. A dependence of ATP proin order to determine activation energies for the hydroduction on the presence of oxalyl phosphate eliminates possible trace contaminating adenylate kinase as the lysis reactions. Activation parameters for the hydrolysis of oxalyl phosphate are collected in Table I. The value source of the activity. The rate varies linearly with the concentration of pyruvate kinase. Progress curves were of AS+ (-0 e. u.) at pH 5.0 and the predominance of Plinear for -1 min, after which product inhibition beO bond cleavage (X30%) suggest that, at this pH, the came apparent. Kinetic constants, K, = 11 f 2 PM and transition state for the hydrolysis has significant dissok cat = 2 min-l, were obtained from a double reciprocal ciative character (22). At pH 1.5, the value of -21 e. u. plot of the initial velocity data (see Fig. 2). Given the for ASS, coupled with a large percentage of C-O bond potent inhibitory properties of the hydrolysis product of cleavage in Hi*0 (>70%) indicates that the predominant oxalyl phosphate, the initial velocities might be subject mechanism of hydrolysis changes to that of an associato inhibition from an oxalate contamination of the subtive nucleophilic attack of water at the carbonyl carbon. strate. 13CNMR, however, showed that the extent of hyThe entropy of activation obtained at pH 12.1 (ASS drolysis of freshly prepared solutions of oxalyl phos= -7 e. u.) is intermediate for associative and dissociaphate that were kept on ice for -30 min was t5%. The initial velocities obtained in assays with fresh preparations of oxalyl phosphate were virtually identical to TABLE I those obtained with oxalyl phosphate from the Dowex Activation Parameters for Hydrolysis of Oxalyl Phosphate” chromatographic treatment. Furthermore, solutions of oxalyl phosphate that had been incubated with oxalate % c-o decarboxylase (40 IU ml-‘) immediately before use gave AH* AS+ bond AG* (kcal/mol) cleavage * initial velocities that were likewise identical to those obat 23°C (kcal/mole) (e.u.) PH tained in the previous experiments. The apparent K,,, for oxalyl phosphate is comparable to the Ki (6 PM) for oxa1.5 22.2 f 1.6 16.0 + 0.9 -21 f 3 >70 5.0 23.2 f 1.0 23.2 + 1.0 0+2 95 present as a trace contaminant in the oxalyl phosphate, is primarily responsible for the low initial velocities. a Error limits represent standard deviations. The reverse reaction (i.e., phosphorylation of oxalate * The percentage of C-O bond cleavage was determined by 31P NMR by ATP) is sufficiently unfavorable, thermodynamically, analysis of Pi formed from hydrolysis of oxalyl phosphate in H,‘sO.
PROPERTIES
OF OXALYL
to complicate characterization. Even in samples that have been treated to minimize bicarbonate content, the inherent bicarbonate-dependent ATPase (14) activity of pyruvate kinase typically produces higher levels of ADP than the equilibrium for the reaction of ATP and oxalate. Moreover, the low equilibrium level of oxalyl phosphate is difficult to detect in the hydroxamate assay. Oxalate and bicarbonate compete for the same binding site on the enzyme (23) such that oxalate effectively inhibits the ATPase activity. Measurements (see Experimental Procedures) of ADP/ATP ratios in assay mixtures of ATP, oxalate, metal cofactors, and enzyme (0.04 to 46 mg ml-‘), which were allowed to equilibrate at room temperature for up to 2 h, showed that the ADP/ATP ratio was co.02 * 0.01. This low apparent extent of reaction could stem from either kinetic or thermodynamic limitations. Significantly longer incubation times were, however, impractical due to the relatively short half-life of oxalyl phosphate under these conditions. Enzyme-catalyzed phosphorylation of oxalate was readily demonstrated in experiments with the P-thio analogue of ATP, ATPPS. Lerman and Cohn (24) demonstrated that the higher y-phospho group transfer potential of ATPPS, relative to ATP, can be used to characterize enzymatic reactions for which equilibrium lies far in the direction of ATP. Assays for ADP/3S/ ATPpS in reaction mixtures of oxalate (10 mM), MgCl, (4 mM), CdClz (1 mM), enzyme (46 mg ml-‘), and Rp ATPpS (3 mM) for 2.5 h, provided an estimate” of an equilibrium constant:
[ATPPSI [OxI [ox _ pl
Ic:!jz, = [ADPpSl
= 2.0 f 0.5 x 102.
Nucleoside phosphorothioates typically react more slowly than their oxynucleotide counterparts (25). The readily measurable extent of reaction of ATPpS and oxalate, under assay conditions similar to those used for the analogous reaction with ATP, suggests that a thermodynamic, rather than a kinetic, factor limits the extent of reaction of ATP and oxalate. The low extent of reaction of ATP and oxalate is consistent with the expected 60-fold ratio of equilibrium constants for reactions of ATP and ATP@ (24). The conditional equilibrium constant, K’$$, for the reaction of ADP@ with Penolpyruvate corrected to pH 7.5 is -170 (24). These results suggest that the phospho group transfer potential of oxalyl phosphate is comparable to that of P-enolpyruvate at pH 7.5. ’ The calculation assumes that the reaction is at equilibrium. Due to the high concentration of enzyme (0.77 mM) relative to the concentrations of substrates, the position of equilibrium may be influenced by differential binding affinities of substrates and products to the enzyme.
43
PHOSPHATE
A
I I 200 Gauss FIG. 3. EPR spectra at 35 GHz for Mn(I1) in solutions of pyruvate, phosphate dikinase, and (A) P-enolpyruvate and oxalate or (B) oxalyl phosphate. Samples contained 20 mM Hepes/NH,OH, pH 6.8,75 mM NH&l, 175 mg ml-’ enzyme, and 0.44 mM MnClx. Sample (A) was and 4.3 mM oxalate generated by addition of 1.7 mM P-&olpyruvate to form the Es-Mn (II)-oxalate complex. Sample (B) was generated by addition of 4 mM oxalyl phosphate to the solution of enzyme and MnC&. The ionic composition of the solution for (B) includes KC1 (-20 mM) that was present in the stock solution of oxalyl phosphate. Spectra were obtained at 2°C.
Substrate properties of oxalylphosphate withpyruuate, phosphate dikinase. The characteristic EPR spectrum for the E,-Mn(II)-oxalate complex (15) provided a sensitive means for detection of phosphorylation of the active site histidyl residue by oxalyl phosphate. Contamination of the oxalyl phosphate by oxalate and phosphate was not a concern in this assay because EPR spectra of the enzyme-Mn(II)-oxalate and enzyme-Mn(II)-Pi species do not resemble the spectrum of the E,-Mn(II)oxalate complex (15). The appearance of this distinctive spectrum for the sample of oxalyl phosphate, enzyme, and Mn(I1) (Fig. 3) indicates that oxalyl phosphate reacts to give oxalate bound to the E, form of the enzyme. Multiple turnovers were not observed with oxalyl phosphate in the direction of formation of Pi and ATP probably due to the unusual stability of the E,-Mn(II)oxalate complex (26). A single-turnover experiment was performed in order to confirm that oxalyl phosphate donated its phospho group to the histidyl residue on the enzyme. A solution containing enzyme and MnCl, was
44
KOFRON AND REED
incubated with oxalyl phosphate. An excess of EDTA was added to sequester Mn(I1) and thereby facilitate dissociation of oxalate from the complex. The resulting mixture was chromatographed over a column of Sephadex G-25 to separate the protein from EDTA and oxalate. Fractions containing protein were pooled and incubated with MnClz, AMP, and PPi. Protein was precipitated by addition of chloroform, and ATP in the supernatant was quantitated by HPLC. Approximately one equivalent (0.95) of ATP was formed per equivalent of dimeric enzyme. Substrate properties of oxalyl phosphate with phosphoenolpyruvate carboxykinase. The activity of oxalyl phosphate with P-enolpyruvate carboxykinase was investigated by a fixed-point assay for formation of GTP. The sample containing oxalyl phosphate and enzyme converted about 40% of the GDP to GTP in 10 min. There was no detectable GTP in experiments with control samples that lacked either enzyme or oxalyl phosphate. Conclusions. Solutions of oxalyl phosphate can be prepared in satisfactory yields by reaction of oxalyl chloride with Pi in aqueous medium. The stability of the compound at neutral pH is sufficient to allow spectroscopic and enzymatic characterization. The acyl phosphate has a high phospho group transfer potential and serves as a phospho group donor in reactions catalyzed by pyruvate kinase, pyruvate, orthophosphate dikinase, and P-enolpyruvate carboxykinase. With pyruvate kinase, however, kcat for oxalyl phosphate reaction with ADP is ~0.02% of that for the corresponding reaction with P-enolpyruvate and ADP. The k,,, for phosphorylation of ADP is also only 1% of the k,,, reported for the oxalate-stimulated positional isotope exchange in ATP (13). Although nonproductive binding can be invoked to account for the low kcat, it is possible that kcat for oxalyl phosphate is limited by release of products. The product complex, enzyme-oxalate-Mn( II)-ATP-Mg( II), is known to have a high affinity for the enzyme (27). The value of kcat for oxalyl phosphate with pyruvate kinase falls into the lower end of the range of constants reported for other analogues of P-enolpyruvate (l-4). ACKNOWLEDGMENTS The authors thank Jenny L. Buchbinder for providing samples of nucleoside phosphorothioates and Dr. Thomas Nowak for the sample of P-enolpyruvate carboxykinase. High-field NMR spectra were ob-
tained at the National Grant RR02301).
Magnetic
Resonance Facility
at Madison
(NIH
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7938-7946. (Colowick, 19. Stadtman, E. R. (1957) in Methods in Enzymology S. P., and Kaplan, N. O., Eds.), Vol. 3, pp. 228-231, Academic Press, San Diego. 20. Jahansouz, H., Mertes, K. B., Mertes, M. P., and Himes, R. H. (1989) Bioorg. Chem. 17,207-216. 21. Lynen, F. (1940) Ber. 73,367-372. 22. Herschlag, D., and Jencks, W. P. (1986) J. Amer. Chem. Sot. 109, 4665-4674. 23. Lodato, D. T. (1986) Ph.D. Thesis, University of Pennsylvania. 24. Lerman, C. L., and Cohn, M. (1980) J. Biol. Chem. 255, 87568760. 25. Cohn, M. (1982) Act. Chem. Res. 15,326-332. 26. Michaels, G., Milner, Y., and Reed, G. H. (1976) Biochemistry 14, 321223219. 26, 224327. Lodato, D. T., and Reed, G. H. (1987) Biochemistry 2250.
