The synthesis and properties in enzymic reactions of substrate analogs containing the methylphosphonyl group

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 19’7, No. 1, October 1, pp. 218-225, 1979

The Synthesis ROBERT

and Properties in Enzymic Reactions of Substrate Containing the Methylphosphonyl Group1

A. LAZARUS,

Department

PATRICIA

of Chemistry, The Pennsylvania

A. BENKOVIC, State University,

AND STEPHEN

Analogs

J. BENKOVIC

University Park, Pennsylvan.ia 16802

Received February 22, 1979; revised April 20, 1979 The properties of the methylphosphonyl group as a substrate analog for the phosphoryl moiety of various biological phosphoryl donors have been investigated in several enzymie phosphoryl transfer reactions. The synthesis and characterization of adenosine 5’-[pmethylphosphonylldiphosphate, adenosine 5’-methylphosphonate, acetyl methylphosphonate, and methylphosphonoenolpyruvate are fully described. Adenosine 5’-[pmethylphosphonylldiphosphate is not a substrate for adenylate kinase, hexokinase, 3-phosphoglycerate kinase, glycerol kinase, phosphofructokinase, creatine kinase, alkaline phosphatase, or nucleoside 5’-diphosphate kinase. Competitive inhibition of ATP was observed with hexokinase and 3-phosphoglycerate kinase with KJK, = 10. Adenosine 5’-methylphosphonate was a substrate for adenylate deaminase and 5’-nucleotidase, but not for adenylate kinase, acid phosphatase, 5’-phosphodiesterase, or 3’-phosphodiesterase. Acetyl methylphosphonate inhibits the reaction of acetyl phosphate with acetate kinase, but methylphosphonoenolpyruvate has no effect upon the reaction of phosphoenolpyruvate with pyruvate kinase. The results indicate that with the exception of 5’-nucleotidase, the methyphosphonyl group is incapable of undergoing phosphoryl transfer. One interpretation among others is that a metaphosphate-type mechanism is required for these processes.

The mechanism of enzymic phosphoryl transfer has been proposed to proceed via either a dissociative monomeric metaphosphate mechanism or an associative nucleophilic addition-elimination process involving the intermediacy of a pentacovalent species. Although model systems have shown that reactions of phosphate monoesters generally proceed via the metaphosphate mechanism whereas phosphate diand triesters react via a nucleophilic pathway, both possibilities must be considered for enzymic phosphoryl transfer, which involves phosphate monoesters as the phosphoryl donor, due to the fact that either protonation or complexation by metal ions may cause a change in mechanism (1, 2). The present study was initiated in order to determine whether the methylphosphony1 group could substitute for the phosphoryl moiety in enzymic phosphoryl transfer reactions. The observations of phos1 This work was supported by Grant GM 13306 from the National Institute of Health.

218

0003-9361-79/110218-03$02.00/o Copyright All rights

0 1979 by Academic Press, of reproduction in any form

phony1 transfer would support an associative mechanism since formation of a monomeric metaphosphate species from the methylphosphonyl group would be disfavored based on the presumption that formation and stabilization of metaphosphate requires two oxyanion ligands to phosphorus (1, 2). Recently Gardner and Byers have shown that a wide range of phosphate analogs including methylphosphonate are substrates for glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) and purine nucleoside phosphorylase (EC 2.4.2.1). Their observation that 3-phosphoglycerate kinase and ADP eliminate product inhibition of the dehydrogenase reaction prompted the suggestion that ATP analogs might be prepared in this manner (3). Previous research on nucleotide analogs has indicated that while modification of the base is tolerated, modification of the phosphate undergoing transfer generally leads to inhibition (4). Modification distant to the phosphate in other phosphoryl donors is also often tolerated in enzymic reactions. We

Inc. reserved.

METHYLPHOSPHONYL

ANALOGS

report in this paper the synthesis of substrate analogs of ATP, AMP, acetyl phosphate, and phosphoenolpyruvate containing the methylphosphonyl group and their properties toward several enzymes that catalyze phosphoryl transfer.

0 A-o-LOA-

0

0

1,‘-0-P &

II ,CH3 \ 0-

A!lTYMe 0 0 II ,CH3 CH3-!-O-P\ 0AcPMe t/m3

A-O-P,

0AMPMe HOOC ' CH3 \ c 0 !' // - - loCH2

MePEP The use of phosphonates as analogs of phosphates has been reviewed recently (5). In general the former are ca. lo-fold more reactive in nucleophilic displacement reactions (6-9); consequently the phosphonates should be active analogs if their intrinsic reactivity were the determining factor. MATERIALS

IN ENZYMIC

REACTIONS

219

available reagent grade chemicals and were generally either distilled or recrystallized before use. Methylphosphonic acid was prepared by hydrolyzing its dimethyl ester in refluxing concentrated HCl overnight according to the method of Kosolapoff (10). After removing the water as the benzene azeotrope, the hygroscopic crystals were recrystallized from ethanol-ether and stored in vucuo over P,O,. Triethylammonium bicarbonate solutions (1 M) were prepared by bubbling CO* through a sintered-glass bubbler into a cooled aqueous solution of the amine until the pH dropped to 7.5. Dowex 5OW-X8, H+, ZO50 mesh was obtained from Bio-Rad and DEAEcellulose (DE 52) from Whatman. Thin layer chromatography was performed on PEI-F* cellulose plates from J. T. Baker. Analytical methods. Ultraviolet spectra were recorded on a Cary Model 118 instrument. A Gilford Model 240 was used for the enzymatic assays. ‘H NMR spectra were recorded on a Varian Associates A-60 spectrometer and 6 values are reported relative to either TMS or DSS. 31P NMR spectra were recorded on a Jeol PS-100FT spectrometer at 40.29 MHz and 6 values are reported relative to 85% H,PCl,; positive values are reported downfield of H,PO,. Determination of the ribofuranoside was determined by sodium metaperiodate oxidation according to the method of Dixon and Lipkin (11). Total phosphate determination was performed by the ashing method of Ames (12). Microanalyses were performed by MHW Laboratories, Phoenix, Arizona. Synthesis of adenosine 5’-[pmethylphosphonyl]diphosphate (ATPyMe). Methylphosphonic acid (192 mg, 2.0 mmol) in 10 ml dry dioxane was converted to the pyridinium, tri-n-octylammonium salt by the addition of pyridine (158 mg, 2.0 mmol) and tri-n-octylamine (707 mg, 2.0 mmol). Diphenyl chlorophosphate (806 mg, 3.0 mmol) and tri-n-butylamine (745 mg, 4.0 mmol) were added and the solution stirred for 3 h at room temperature. The solution was evaporated under vacuum; 20 ml ether and 40 ml petroleum ether were added, and the mixture was left at 4°C for 30 min. The supernatant was decanted and the residue dissolved in 10 ml dioxane and evaporated to dryness. ADP.Nk.2.5 H,O (532 mg, 1.0 mmol) was dissolved in 50 ml methanol-water (50%) and was converted to the pyridinium salt by passage (0.75 ml/min) through a 50 x l-cm column of Dowex W-X8 that had been converted to its pyridinium form by addition of an aqueous

AND METHODS

Materials. Diphenyl chlorophosphate, dicyclohexylcarbodiimide, and dimethyl methylphosphonate were obtained from Aldrich Chemical Company. Adenosine, adenosine 5’-diphosphate (disodium salt), phosphoenolpyruvate (cyclohexylammonium salt), NADH, and bromopyruvic acid were obtained from Sigma. Dimethyl methylphosphonite was purchased from Orgmet, Inc. Other materials were commercially

* Abbreviations used: ATPyMe, adenosine 5’-[pmethylphosphonylldiphosphate; AMPMe, adenosine 5’-methylphosphonate; MePEP, methylphosphoneonolpyruvate; AcPMe, acetyl methylphosphonate; PEI, polyethyleneimine; FATP, y-fluoroadenosine 5’-triphosphate; E, enzyme; P, phosphoryl; TMS, tetramethylsilane; DSS, 2,2-dimethyl-2-silapentane5-sulfonate.

220

LAZARUS,

BENKOVIC,

pyridine solution to the free acid form followed by washing the resin with water to neutrality, then equilibrating with 50% methanol-water. The effluent was evaporated under vacuum using a mechanical pump and the residue then dissolved in 20 ml of methanol. Tri-n-butylamine (189 mg, 1.0 mmol) and tri-n-octylamine (353 mg, 1.0 mmol) were added and the solution stirred for 0.5 h. The solvent was evaporated and the contents dried by repeated addition and evaporation of three 5-ml portions of dry pyridine. The resulting oil was dissolved in 10 ml dry pyridine and added to the residue containing the diphenylphosphor-y1 methylphosphonate. After stirring at room temperature for 2 h the solution was evaporated to a yellow oil. The subsequent steps were performed at room temperature without delay. The residue was extracted with ether and the aqueous layer was applied to a 3.0 x 22-cm DEAE-cellulose column and eluted with a linear gradient derived from solutions 0.05 and 0.4 M in triethylammonium bicarbonate (pH 7.5, 1.25 1 each). The results of the separation are shown in Fig. 1. The four different fractions were pooled and each was evaporated at 30°C using a mechanical pump. Residual triethylammonium bicarbonate was removed by four evaporations with loml portions of methanol. The residue for each fraction was transferred to a 40-ml centrifuge tube and dissolved in 5 ml methanol and 15 ml acetone. A solution of 1 M NaI in acetone was added dropwise until no more precipitate formed. The precipitate was collected by centrifugation, washed three times with 15 ml acetone and finally with 15 ml ether. The ether was decanted and the white crystals of the fractions were dried under a flow of nitrogen. The

AND BENKOVIC isolated yields of fractions 2,3, and 4 were 311,60, and 49 mg, respectively. Fraction 1 was not analyzed. Thin-layer chromatography of PEI-F cellulose plates with 1 M NaCl gave the following R, values: adenosine, 0.58, AMP, 0.45; ADP, 0.13; ATP, 0.06; fraction 2, 0.12, and 0.38; fraction 3, 0.05, 0.11, and 0.38; fraction 4, 0.24. 31P NMR (D,O) of fraction 2 showed a doublet (J = 21.5 Hz) at +17.9 ppm and a peak at -23.5 ppm along with several peaks at -10.8 and - 11.5 ppm which is consistent with a mixture of ADP and ATPyMe. Fractions 2,3, and 4 were analyzed for ADP by adding ca. 0.03 mg to a l-ml solution composed of 80 mM triethanolamine pH 7.6, 0.5 mM phosphoenolpyruvate, 65 mM KCl, 20 mM MgC12, 0.2 mM NADH, 21 units/ml lactic dehydrogenase, and 10 units/ ml pyruvate kinase. The results showed that fraction 2 contained 40% ADP while fractions 3 and 4 contained less than 3% ADP. Fraction 2 was purified in the following manner. A lo-ml solution containing 70 mM triethanolamine pH 7.6, 100 mM KCl, 20 mM MgCl*, 11.2 mM phosphoenolpyruvate, 100 mg fraction 2, and 50 units of pyruvate kinase was monitored by tlc(PEI-F, 1 M NaCl). The residual ADP was converted to ATP within 5 min while ATPyMe was unaffected. The enzyme was denatured by shaking the solution with 5 ml of chloroform. The aqueous layer was separated and the chloroform layer extracted several times with water. The combined aqueous extracts (ea. 50 ml) were filtered through glass wool and chromatographed on the same DEAE-cellulose column as before using a O.l0.4 M linear gradient of triethylammonium bicarbonate at pH 7.5 (see inset of Fig. 1). The elution profile showed the presence of ATPyMe (eluted at 0.20 M

1.5 -

20

40

60

so 130 120 TUBE NUMBER

140

160

FIG. 1. Chromatographic purification of ATPyMe. Conditions: room temperature; flow rate 2.4 ml/ min of a linear gradient of 0.05-0.40 M triethylammonium bicarbonate buffer pH 7.5; 14.4-ml fractions collected. The inset shows the rechromatography (0.10-0.40 M triethylammonium bicarbonate buffer pH 7.5; 1.4 ml/min; 11-ml fractions collected) of ATPyMe and ADP (fraction 2) after treatment with pyruvate kinase. The columns were monitored by pipetting 0.1 ml of eluent into 2 ml H,O and reading &b-O.

METHYLPHOSPHONYL

ANALOGS

R, 0.38) and ATP (eluted at 0.25 M, R, 0.06). The solution of ATPyMe was treated as before and the sodium salt obtained as pure white crystals was stored at 0°C in a desiccator.3 The total yield of ATPyMe was 140 mg (21% based on a molecular weight of 668 and 28% if recovered ADP is taken into account). Assuming an extinction coefficient of 15,400 at 259 nm the molecular weight is 668 which corresponds to ATPyMe . 3Na.5.5 H20. The ratio of adenine:ribose:totaI phosphate was 1.0:0.99:3.02. 31P NMR (9 mg/ml D,O [Z mM EDTA]) 6 +17.8 ppm (d, Jpp = 22.5 Hz, CH,-P,), - 11.4 ppm (d, J, = 20.2 Hz, P,), -23.1 ppm (d of d, Jpp = 22.6 Hz, Pp). The coupled 31P NMR spectrum was also run and gave identical values for P, and Pp, however P, was a doublet of quartets with J,, = 23.0 Hz and JPH = 17.1 Hz. Alternatively fraction 2 was purified by removing the ADP with alkaline phosphatase (Worthington, chicken intestine, 2.6 units/mg). A solution containing 4 mg of fraction 2 was incubated with 0.8 mg of alkaline phosphatase in 0.05 M NaHC03, pH 8.2 and was monitored over 4 h by tic (1 M NaCI). The contaminating ADP was first hydrolyzed to AMP and then to adenosine whereas the ATPyMe remained intact. Synthesis (AMPMe).

of

adewosine-5’-methylphosphonate

This was prepared by the method of Myers et al. (13) to give a 69% yield of white crystals; mp 187°C (dec.) (lit, 180-181°C dec.). The product showed a single spot on tic (1 M NaCl on PEI-cellulose) R, = 0.74. ‘H NMR (D,O) showed a doublet at F 1.33 ppm withJ = 16 Hz, which corresponds to the methylphosphonyl group, as well as resonances for adenosine. Synthesis of cyclohexylammonium hydrogen methylph~osphonoenolpyruvate (MePEP). This compound was prepared by a Perkow reaction using a procedure analogous to the synthesis of PEP by Stubbe and Kenyon (14). A solution of bromopyruvic acid (0.835 g, 5.0 mmol; freshly recrystallized from CHCI,) in 10 ml Et,0 was added dropwise to an ice bath cooled solution of freshly distilled dimethyl methylphosphonite (0.57 g, 5.3 mmol) in 10 ml Et,0 over 10 min. The cloudy solution that formed cleared within 5 min and stirring was continued at room temperature for 1 h. The solution was rotary evaporated to give a clear pale yellow oil which was hydrolyzed in 10 ml Hz0 for 1 h. Cyclohexylamine (0.495 g, 5.0 mmol) was added and the solution was rotary evaporated to a yellow oil which was taken up in 5 ml MeOH and 40 ml Et,0 to give 194 mg of off-white crystals. Recrystallization from EtOH-Et,0 afforded 143 mg 3 When this purification scheme was repeated on a larger scale, a small amount of ADP still remained, possibly due to the fact that ATP is an inhibitor of pyruvate kinase at high concentrations. If this occurred, the phosphorylation of ADP was repeated again and rechromatographed until the ATPyMe was free of ADP as monitored, both by tic and enzymically.

IN ENZYMIC

221

REACTIONS

(11% yield) of pure white crystals; mp 151-152°C (dec.); ‘H NMR (D,O) S 1.1-2.2 (br, 11 H, cyclohexyl H), 1.41 (d, J = 17 Hz, 3H, CH,P), 2.7-3.5 (br, 2H, NH,), 5.48 (apparent t, J = 2 Hz, lH, vinyl H), 5.85 (apparent t, J = 2 Hz, lH, vinyl H); ir (KBr) 3400 br, 3200-2300 br, 1’710 s, 1620 m, 1190 s, 900 m cm-l. Anal. Calcd. for &,H,,NO,P: C, 45.29; H, 7.60; i, 5.28; P, 11.68. Found: C, 45.14; H, 7.79; N, 5.13; P, 11.89. Synthesis (AcPMe).

of

lithium

acetyl

methylphosphonate

Methylphosphonodichloridate was prepared by refluxing methylphosphonic acid (12 g, 0.125 mol) in 40 ml of thionyl chloride for 3 h and stirring at room temperature overnight. After removal of excess SOCI,, the product was distilled at 85°C at 54 mm Hg to give 14.34 g (86% yield) of a clear oil which crystallized, mp 34°C (lit. 33°C (15)). ‘H NMR (Ccl,) 6 2.59 (d, J = 17 Hz, CH,P). Diacetyl methylphosphonate was prepared by the dropwise addition of 2.66 g (0.02 mol) methyl phosphonodichloridate in 50 ml Et20 to a stirred suspension of 8.35 g (0.05 mol) silver acetate in 100 ml Et,0 over 20 min. The latter had been freshly prepared by addition of NaOAc.3H20 (4.1 M) to AgNOB (4.0 M) in H20, followed by filtration of the white precipitate, washing with HzO, EtOH, and Et*O, and drying at 100°C for 1 h. After stirring at room temperature overnight in the dark, the mixture was filtered and the filtrate rotary evaporated to give 3.22 g (89% yield) of a pale yellow oil. ‘H NMR (CDCI,) showed a methylphosphonyl peak and an acetyl methyl peak along with several small impurities. Attempted distillation of the crude product failed. The crude diacetyl methylphosphonate (0.72 g, 4.0 mmol) and LiOH (0.096 g, 4.0 mmol) in 3.5 ml H,O were combined and kept cold for 30 min (pH = 3.4). This solution was added to 100 ml acetone, cooled, and 250 mg (44%) fine white crystals were collected. ‘H NMR (D,O) S 1.53 (d, J = 16 Hz, 3H, CH,P), 2.22 (s, 3H, CH,Q4 =P NMR (H,O) 6 24.25 (s, 72.8%) 17.03 (s, 27.2%). Conversion to the hydroxamate revealed that 76.3% of the crude salt was reactive which is in agreement with the NMR data that the compound obtained is ca. 75% pure (16). Enzymes and assays. The enzymic reactions of the substrate analogs were assayed by tic and/or a specific enzymie spectrophotometric assay. The following enzymes were purchased from Sigma; the specific assay conditions are referenced after the enzyme: acetate kinase, EC 2.7.2.1 (17); adenylate kinase, EC 2.7.4.3 (18); 3-phosphoglycerate kinase, EC 2.7.2.3 (3, 17); hexokinase EC 2.7.1.1 (17); nucleoside 5’-diphosphate kinase, EC 2.7.4.6 (17); creatine kinase, EC 2.7.3.2 (18); pyruvate kinase, EC 2.7.1.40 (17); adenylate deaminase, EC 3.5.4.6 (19); snake venom phospho* An impurity appears at 1.25 (d, 1.5 H) which is probably methylphosphonic acid and corresponds to 33% of the total.

222

LAZARUS,

BENKOVIC,

die&erase, EC 3.1.4.1 (18) for inhibition experiments; and calf spleen phosphodiesterase, EC 3.1.4.18 (18). The following enzymes were assayed as follows: glycerol kinase, EC 2.7.1.30: 70 mM triethanolamine, pH 7.6, ‘7.1 mM MgCl,, 7.1 mM glycerol, 5.7 mM ATP or ATPyMe, and 23 units of glycerol kinase by tic; phosphofructokinase, EC 2.7.1.11: 40 mM Tris, pH 8.0, 50 mM KCl, 5 mM MgCl*, 1 mM EDTA, 0.1 mM dithiothreitol, 0.4 mM KH,PO,, pH 8.0,0.5 mM phosphoenolpyruvate, 0.17 mM NADH, varying amounts of ATP and/or ATPyMe, 0.1 mM fructose &phosphate, 8 units pyruvate kinase, 16 units lactic dehydrogenase, and varying amounts of phosphofructokinase at 340 nm; alkaline phosphatase, EC 3.1.3.1: 0.05 M NaHCO,, pH 8.2, 3.8 mM ATPyMe, and 2 units of alkaline phosphatase by tic; acid phosphatase (wheat germ), EC 3.1.3.2: 0.1 M sodium acetate, pH 5.0, 50 mM AMP or AMPMe, and 1 unit of acid phosphatase by tic; snake venom phosphodiesterase, EC 3.1.4.1: 0.17 M Tris, pH 8.9, 10 mM MgCl,, 0.25 unit of adenosine deaminase, 0.1 mM AMPMe, and 0.2 unit of the 5’-phosphodiesterase at 265 nm for substrate activity; 5’nucleotidase, EC 3.1.3.5: 0.25 M Tris, pH 7.5,0.25 unit of adenosine deaminase, and varying amounts of AMPMe and 5’-nucleotidase at 275 nm. Reagents for the individual assays were the highest grade of commercially available chemicals. The tic method involved following the reaction over the course of several hours by following the nucleotide products on PEI-F cellulose plates using 1 M NaCl as the developing solvent. Typically 0.01-0.02 pmol of nucleotide was applied to the tic plate so that a reaction that has proceeded ea. 3% to completion would be detectable. High concentrations of both substrate analog and enzyme were used. The natural substrate was run as well and the product developed on the same tic plate as the substrate analog reactions. RESULTS

AND DISCUSSION

Enxymic Reactions of ATPyMe

The reactions of various ATP kinases utilizing the appropriate phosphate acceptor and ATPyMe as the phosphate donor were monitored by ion-exchange thin layer chromatography and/or a specific spectrophotometric assay in order to determine if ATPyMe is a substrate. The tic method was used for adenylate kinase, hexokinase, nucleoside 5’-diphosphate kinase, glycerol kinase, creatine kinase, and alkaline phosphat.ase; the spectrophotometric method for hexokinase, 3-phosphoglycerate kinase, phosphofructokinase, adenylate kinase, and nucleoside 5’-diphosphate kinase. The concentrations of all of the substrates were

AND BENKOVIC

saturating to achieve maximal enzyme activity. In the case of creatine kinase where phosphoryl transfer is thermodynamically unfavorable (K = 10-9, pyruvate kinase, phosphoenolpyruvate, and lactic dehydrogenase were added to pull the reaction to completion (18). The reactions employing ATP (R, = 0.06) as substrate indicated the formation of ADP (R, = 0.13) as product within several minutes whereas for ATPyMe (R, = 0.38), no indication of any reaction was observed over several hours. A turnover rate of ca. 10e2 min-’ would have been detected. Since ATPyMe is not a substrate for these enzymes, inhibition studies were carried out to determine if it is bound to the enzyme. A Dixon plot for the reaction ATPyMe with hexokinase is shown in Fig. 2 which clearly indicates competitive inhibition toward ATP.5 The ratio of Ki(ATPyMe)I K,(ATP) = 13.3. Similarly competitive inhibiton was observed with 3-phosphoglycerate kinase with KiIK, = 10.7 (Fig. 3).5 The utilization of ATP by phosphofructokinase was also inhibited threefold by ATPyMe (ATP = 7 PM; ATPyMe = 0.8 mM), however with nucleoside 5’-diphosphate kinase, whose reaction proceeds through the formation of a histidine-phosphate bound as an E-P intermediate (21), no inhibition was observed at ATP = 0.1 mM and ATPyMe = 1.3 mM. The results with the ATPyMe analog are in agreement with those of several other nucleoside analogs. y-Fluoroadenosine 5’-triphosphate (FATP) was not a substrate for myosin, heavy meromysin, hexokinase, or E. coli alkaline phosphatase (22). Similarly, GTP analogs modified on the terminal phosphate were not hydrolyzed by elongation factor G and ribosomes, but were competitive inhibitors of the ribosome-dependent EF-G GTPase (23). At physiological pH values ATPyMe is completely dissociated (ATPyMe3-), whereas ATP exists as HATP3- or ATP*(24). Since ATP*- binds Mgz+ ca. 40- to 80fold more strongly than HATP3- or FATP3(22), the decreased reactivity of ATPyMe 5 Since mixed (noncompetitive) inhibition may resemble competitive inhibition, the data were also plotted using Lineweaver-Burk plots which contlrmed that inhibition is competitive (20).

METHYLPHOSPHONYL

ANALOGS

FIG. 2. Dixon plot showing the effects of ATPyMe on the reaction of ATP with glucose catalyzed by hexokinase. The assays were run as follows: 80 mM triethanolamine, pH 7.6, 220 mM glucose, 6 mM MgC&, 0.66 mM NADP, 0.5 unit of glucose 6-phosphate dehydrogenase, 0.02 unit of hexokinase, and varying amounts of ATP (0.04 mM, (m) 0.05 mM (@), or 1.94 mM (A) and ATPyMe in a O.&ml assay. Values for Ki (ATPyMe) and K, (ATP) are 2.0 and 0.15 mM, respectively.

may be partially attributed to a lower concentration of the important Mg2+-ATPyMe3complex since the Mg:ATPyMe ratio was ca. 1:l experimentally. However this rationale does not adequately explain the apparent total absence of analog reactivity. Recently Viola and Cleland have demonstrated that yeast hexokinase can utilize both Mg2+-HATP3and Mg2+-ATP4equally well (25). The suggestion that methylphosphonate

-6

-4

-2

0 2 4 6 cATP+4*,, rnM

6

10

FIG. 3. Dixon plot showing the effects of ATPyMe on the reaction of ATP with glycerate 3-phosphate catalyzed by 3-phosphoglycerate kinase. The assays were run as follows: 70 mM triethanolamine, pH 7.6, 5.6 mM glycerate 3-phosphate, 0.18 mM NADH, 0.8 mM EDTA, 5 mM MgSO,, 1.7 units of glyceraldehyde 3-phosphate dehydrogenase, 0.016 unit of 3-phosphoglycerate kinase, and varying amounts of ATP (0.19 mM (m), 0.38 mM (O), and 9.72 mM (A)) and ATPyMe in a 0.5-ml assay. Values for K, (ATPyMe) and Km (ATP) are 2.9 and 0.27 mM, respectively.

IN ENZYMIC

REACTIONS

223

could be used to synthesize ATPyMe through the glyceraldehyde 3-phosphate dehydrogenase-3-phosphoglycerate kinase coupled pathway is in direct cont.rast to our results and violates the law of microscopic reversibility. To resolve the dilemma we repeated the procedure of Gardner and Byers, following the reaction spectrophotometrically, and isolated the products by chromatography on DEAE-cellulose (3). No ATPyMe was isolated under conditions where it was shown to be stable. At present we have no explanation for the reported removal of the product inhibition of the dehydrogenase reaction by the addition of kinase, since the ADP does not become phosphorylated to form ATPyMe. The K,(CH3P032-)/K,(HOP032-) was found to be 9.5 for the dehydrogenase reaction (3). It is interesting to note that replacement of oxygen by a methyl group lowers the apparent binding constant by a factor of ea. 10 regardless of whether the phosphate is a substrate or an inhibitor. Enzymic

Reactions

of AMPMe

Some of the primary enzymatic reactions utilizing AMP involve phosphorylation to give ADP, hydrolysis to give adenosine, and deamination of the purine to give IMP. AMPMe is a substrate for 5’-adenylic acid deaminase with a K, = 3.9 KIM at pH 6.3 (K,(AMP) = 0.4 mM>. This result is not unexpected in view of the broad substrate specificity of this enzyme (19). The methyl group again causes a lo-fold decrease in the apparent binding constant. The reaction of AMPMe (R, = 0.74) was studied with adenylate kinase and ATP to see if the methylphosphonyl group can act as a phosphoryl acceptor. The reaction was monitored by tic for the appearance of ADP and ADPaMe6 over 3 h, however no reaction occurred. The formation of ADP utilizing AMP as the substrate was detected under identical conditions within 5 min. A spectrophotometric assay using the pyruvate kinase-lactic dehydrogenase couple showed no inhibition with AMPMe = 2.5 6 ADPaMe may be hydrolytically unstable, however the adenylate kinase could function as an ATPase.

224

LAZARUS,

BENKOVIC,

and AMP = 0.03 mM. Inhibition of adenylate kinase with ATPyMe = 1.5 mM and ATP = 0.015 mM resulted in only a threefold decrease in rate. The attempted hydrolysis of AMPMe by calf spleen phosphoacid phosphatase, diesterase (3’), or snake venom phosphodiesterase (5’) was monitored by tic. No reaction was detected. AMPMe was a competitive inhibitor (Ki = 1.8 InM, pH 8.9) toward bis p-nitrophenyl phosphate for the snake venom enzyme. However, AMPMe was a substrate for the phosphomonoesterase, 5’-nucleotidase (K, = 0.13 mM, pH 7.5) with a V = 0.05 V for AMP. Hol$ and co-workers have observed that nucleoside 5’-0-hydroxy- and aminomethanephosphonates are resistant to alkaline phosphatase and snake venom phosphodiesterase, but are substrates for the snake venom 5’-nucleotidase (26-28). InM

BENKOVIC

AND

Enzymic Acetate

Reaction Kinase

of AcPMe

with

The results of AcPMe as a substrate analog for acetyl phosphate indicate that while no phosphorylation of ADP was detected, AcPMe does inhibit the reaction of acetyl phosphate with acetate kinase. A fourfold decrease in rate was observed when acetyl phosphate = 10 mM and AcPMe = 20 111M. Earlier evidence pertaining to the mechanism of acetate kinase has indicated the presence of an E-P intermediate through a carboxylate function (31), although more recent experiments rule against this postulate (R. A. Lazarus and S. J. Benkovic, unpublished results; J. Knowles, personal communication). The fact that acetyl phosphate is still active indicates that AcPMe does not phosphorylate the enzyme to form a dead-end complex. CONCLUSION

Enxymic Reaction of MePEP Pyruvate Kinase

with

A large number of analogs of phosphoenol pyruvate have been prepared and their enzymic reaction studied with pyruvate kinase (14, 29, 30), however modifications have primarily been distal to the phosphate. Indications are that an ionized carboxylate group is necessary for good binding and reactivity and the bulk tolerance at the 3-position of PEP is limited. Replacement of the enol oxygen with a methylene group gives a phosphonate incapable of phosphoryl transfer, however its inhibitory effect toward pyruvate kinase is relatively small. The reaction of MePEP, a phosphonate that is capable of phosphoryl transfer, with pyruvate kinase as followed by tic indicated that no phosphorylation of ADP had taken place after 18 h under conditions where 3% reaction could be detect.ed and where PEP reacts within several minutes. At MePEP = 10 InM and PEP = 0.1 mM no inhibition was detected. The fact that MePEP does not bind to pyruvate kinase may be due to either steric bulk at the phosphorus or its inability to complex with the required metal ions at. the active site owing to the decreased basicity of the phosphoryl moiety.

The results presented in this paper indicate that the methylphosphonyl group is incapable of undergoing enzymic phosphoryl transfer with the exception of AMPMe with 5’-nucleotidase. While this supports a dissociative metaphosphate mechanism, steric or metal binding effects may be operative such that the configuration of the phosphoryl donor is changed at the active site. There seems to be no general pattern of interaction of these analogs with the enzymes studied, however inhibition when detected was competitive with Kil K, = 10. While both negatively charged oxygens are necessary for the compounds to act as substrates, binding to the enzyme may only require one. Spectroscopic studies are underway in an attempt to determine the configuration of the bound analog. REFERENCES

1. BENKOVIC, S. J., AND SCHRAY, K. J. (1973) in The Enzymes (Bayer, P. D., ed.), Vol. VIII, pp. 201-238, Academic Press, New York. 2. BENKOVIC, S. J., AND SCHRAY, K. J. (1978) in Transition States of Biochemical Processes (Gandour, R. D., and Schowen, R. L., eds.), pp. 493-527, Plenum, New York. 3. GARDNER, J. H., AND BYERS, L. D. (1977) J. Biol.

Chem.

250, 5925-5927.

METHYLPHOSPHONYL

ANALOGS

4. YOUNT, R. G. (1975) in Advances in Enzymology (Meister, A., ed.), Vol. 43, pp. l-56, Wiley, New York. 5. ENGEL, R. (1977) Chem. Rev. 77, 349-367. 6. BEHRMAN, E. J., BIALLAS, M. J., BRASS, H. J., EDWARDS, J. O., AND ISAKS, M. (1970) J. Org. Chem. 35, 3063-3069. 7. BEHRMAN, E. J., BIALLAS, M. J., BRASS, H. J., EDWARDS, J. O., AND ISAKS, M. (1970) J. Org. Chem. 35, 3069-30’75. 8. KIRBY, A. J., AND YOUNAS, M. (1970) J. Chem. Sot. (B), 510-513. 9. KIRBY, A. J., AND YOUNAS, M. (1970) J. Chem. Sot. (B), 1165-1172. 10. KOSOLAPOFF, G. M. (1954) J. Chem. Sot., 32223225. 11. DIXON, J. S., AND LIPKIN, D. (1954) Anal. Chem. 26, 1092- 1093. 12. AMES, B. N. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. VIII, pp. 115- 118, Academic Press, New York. 13. MYERS, T. C., NAKAMURA, K., AND DANIELZADEH, A. B. (1965) J. org. C&m. 30, 15171520. 14. STUBBE, J. A., AND KENYON, G. L. (1972) Biochemistry 11, 338-345. 15. KINNEAR, A. M., AND PERREN, E. A. (1952) J. Chem. Sot., 3437-3445. 16. GARDNER, C. R., LANGER, R. S., AND COLTON, C. K. (1976) Anal. Biochem. 76, 654-656. 17. Biochemica Information (1973) Vol. I, Boehringer Mannheim GmbH, Biochemica.

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18. Biochemica Information (1975) Vol. II, Boehringer Mannheim GmbH, Biochemica. 19. ZIELKE, C. L., AND SUELTER, C. H. (1971) J. Biol. Chem. 246, 1313-1317. 20. PURICH, D. L., AND FROMM, H. J. (1972) Biothem. Biophys. Acta 268, 1-3. 21. PARKS, R. E., JR., AND AGARWAL, R. P. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. VIII, pp. 307-333, Academic Press, New York. 22. HALEY, B., AND YOUNT, R. G. (1972) Biochemistry 11, 2863-2871. 23. ECKSTEIN, F., BRUNS, W., AND PARMEGGIANI, A. (1975) Biochemisty 14, 5225-5232. 24. BRUICE, T. C., AND BENKOVIC, S. J. (1967) Bioorganic Mechanisms, Vol. 2, pp. 153-180, Benjamin, New York. 25. VIOLA, R. E., AND CLELAND, W. W. (1978) Biochemistry 17, 4111-4117. 26. GULYAEV, N. N., AND Hoty, A. (1972) FEBS Lett. 22, 294-296. 27. HOLY, A., AND HONG, NG. D. (1971) Collect. Czech. C&m. Commun. 36, 316-317. 28. HoLP, A., AND HONG, NG. D. (1972) Collect. Czech. Chem. Commun. 37, 2066-2076. 29. STUBBE, J. A., AND KENYON, G. L. (1971) Biochemisty 10,2669-2677. 30. WOODS, A. E., O’BRYAN, J. M., MUI, P. T. K., AND CROWDER, R. D. (1970) Biochemistry 9, 2334-2338. 31. ANTHONY, R. S., AND SPECTOR, L. B. (1972) J. Biol. Chem. 247, 2120-2125.