the mechanism of enzymic phosphate transfer reactions* of agricultural

THE MECHANISM OF ENZYMIC PHOSPHATE TRANSFER REACTIONS*

BY W. H. HARRISON, P. D. BOYER, AND A. B. FALCONE

(From the Department of Agricultural Biochemistry, University of Minnesota, St. Paul, Minnesota)

(Received for publication, October 12, 1954)

Various experimental approaches may yield information to make possible a better understanding of enzymic phosphate transfer. Essential at the outset is a clear recognition of the substrates and cofactors, if any, involved ; these have been relatively well defined for many enzymic phosphate transfer reactions. With the exception of Mg++, which appears necessary for all enzymic phosphate transfer reactions studied (1, 2), spe- cial cofactor requirements have been demonstrated for only a few phosphate-transferring enzymes, as illustrated by the requirement of pyruvate kinase for K+ (3) and of phosphoglucomutase for glucose-l ,6-diphosphate

(4). Valuable information about phosphate transfer may be obtained with

use of isotopes. For example, Meyerhof et al. showed that Ps2-labeled inorganic phosphate was not incorporated into hexose monophosphates during their enzymic interconversion (5), and Jagannathan and Luck showed incorporation of substrate phosphate into phosphoglucomutase (6). The suggestion of the latter studies that an enzyme-phosphate intermediate participates in the catalysis has beeh supported by later results (7). The value of 01* for study of enzymic reactions involving phosphate has been shown by the excellent researches of Cohn (8, 9).

In this paper are presented results of studies with use of 018 to determine the fate of phosphate oxygen in enzymic phosphate transfer reactions and in a non-enzymic phosphate transfer reaction together with additional results from study of the pyruvate kinase reaction with pyruvate-2-04. From these and other studies, postulates about, the mechanism of enzymic phosphate transfer are made.

Materials and Methods

Enzyme Preparations-The rabbit muscle extract used for the experiments reported in Table I was prepared by grinding 30 gm. of frozen,

* Taken in part from portions of the thesis of W. H. Harrison for the degree of Doctor of Philosophy. Supported by a research grant from the National Science Foundation and a research grant (1033) from the National Institutes of Health,

Public Health Service. Paper No. 3243, Scientific Journal Series, Minnesota Agri-

cultural Experiment Station.

303

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304 PHOSPHATE TRANSFER REACTIONS

minced rabbit muscle with two 8 ml. portions of cold 0.03 N KOH. The ground mixture was pressed through folded gauze to give a turbid extract.

The acetone powder of a rat muscle extract was prepared as described by Boyer et al. (3). The acetone powder of a yeast extract was prepared essentially by the procedure of Hochster and Quastel (10) as described elsewhere (11).

The pyruvate kinase source was the fraction which precipitates at 72 per cent saturation with ammonium sulfate at pH 7.5 in the procedure of Cori et al. for the preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (12). The acetokinase used was a purified preparation from Escherichia co&.’ The alkaline phosphatase was a commercial preparation (Armour’s intestinal phosphatase).

Isotope - Labeled CompoundsPyruvamide - 2 - Cl4 was purchased from Tracerlab, Inc., Boston, Massachusetts; the amide was hydrolyzed by al- lowing a solution to stand for several hours in 0.1 N HCl at room temperature.

Inorganic orthophosphate (Pi) labeled with O’* was prepared by careful treatment of PZOG with Hz018 (Stuart Oxygen Company, San Francisco, California) and isolation of the phosphate as KHzPO, was done essentially as described by Cohn (8).

3-Phosphoglycerate labeled with 0’8 in the -O- oxygen9 of the phosphate was prepared by the action of a yeast extract on glucose in the presence of Pi-O”. Glucose was converted to hexose diphosphate in ac- cordance with the Harden-Young equation (13) by incubating at room temperature a mixture containing 600 mg. of yeast acetone powder, 8 mg. of diphosphopyridine nucleotide (DPN), 40 pmoles of MgCL, 20 pmoles of adenosine triphosphate (ATP), 2 mmoles of Pi-O18 (0.425 atom per cent excess 018), 3 mmoles of glucose, and 2 mmoles of acetaldehyde in a total volume of 18 ml. at pH 6.2. After 1 hour’s incubation no Pi and 1.35 mmoles of fructose-l ,6-diphosphate were present. Then 2 ml. of 0.05 M

IW and 2 ml. of 0.02 M sodium arsenate, pH 6.2, were added. After an additional 75 minutes incubation, 100 mg. of yeast acetone powder were added; after another 45 minutes incubation 0.26 mmole of fructose-l ,6-

1 The acetokinase was kindly made available for these studies by Professor S.

Ochoa and Dr. I. Rose of New York University.

ii 2 In a compound such as R-O-P-O-, a principal resonant form of which is

O- I

I 0 R-0-P+-O-, the 3 oxygens not bound to the R group are alike and may be con-

I O-

veniently designated as the -O- oxygens of the phosphate.

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W. H. HARRISON, P. D. BOYER, AND A. B. FALCONE 305

diphosphate remained. The reaction was stopped by the addition of 1 ml. of 30 per cent trichloroacetic acid and the mixture was chilled and centrifuged.

The supernatant solution was neutralized to pH 8.2 with KOH, first with saturated then 1 N KOH. To the solution 25 per cent barium acetate was added carefully until no additional precipitate was formed. The solution was chilled; the precipitate was collected by centrifugation and suspended in 18 ml. of 0.1 N HCI. The undissolved residue was removed by centrifugation and discarded. On standing overnight in a refrigerator, crystals of barium phosphoglyeerate formed. The crystals were collected by filtration and dried in a vacuum desiccator; the yield was 101 mg. The crystals contained no inorganic phosphate, fructose-l ,6-diphosphate, or easily hydrolyzable phosphate. The total phosphorus present was 9.0 per cent; calculated for BaOOCCHOHCH20P03H.2Hz0, 8.6 per cent; for the monohydrate, 9.1 per cent.

For determination of the 018 content of the phosphate group, 5 ml. of a solution at pH 9.3 containing 59 pmoles of the Ol*-labeled S-phosphoglycerate, 0.06 no MgC12, 0.07 M NHz-NH,+ buffer, and 4 mg. of an intestinal alkaline phosphatase preparation were incubated at 37.5” for 75 minutes. The hydrolysis was 77 per cent complete in this time. The MgNH4P04 formed was collected and after addition of carrier inorganic phosphate the 0’8 content was determined as described later. The OIS content was equivalent to the presence of 0.244 atom per cent excess 0’8 in the -O- phosphate oxygens of the original 3-phosphoglycerate. From the mechanism of the enzymic reactions involved, excess O’* would be expected only in the -O- oxygens of the phosphate group and not in the oxygen of the C-O-P linkage. Only the -O- oxygens would appear in the Pi formed by alkaline phosphatase hydrolysis, because phosphatases catalyze cleavage of the O-P linkage (8).

Alkaline phosphatase catalyzes a slow exchange of phosphate oxygen with that of mater (14). To minimize this possibility, the reaction conditions for the above experiment were such as to precipitate the Pi formed as MgNHkP04. As a further check, an experiment was run in which Pi labeled with 018 replaced the 3-phosphoglycerate; the Pi showed no loss of excess Ols.

Isotopic Analyses for C14-The relative activity of preparations containing Cl4 was determined by conventional techniques Sth an internal Geiger gas flow counter; samples were counted to within 2 per cent standard error. For isolation of phosphopyruvate-2-C’ in the presence of large excesses of pyruvate-2-C”, the pyruvate was converted to the 2,4-dinitrophenylhydrazine derivative and the phosphopyruvat,e (together with other Ra-insol- uble phosphates) was isolated as the Ba salt. A typical isolation was as follows: To a 1 ml. enzyme reaction mixture was added 0.5 ml. of 0.01 M

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pyruvate (to dilute pyruvate already present) followed by 1 ml. of 0.25 per cent 2,4-dinitrophenylhydrazine in 2 N HCl. The mixture was allowed to stand for 10 minutes at 27”. Then 5 ml. of 95 per cent ethanol were added, the contents were mixed, and 1.5 ml. of 3 M sodium acetate and 0.2 ml. of 0.4 M barium acetate were added. The contents were well mixed, then allowed to stand for 15 to 20 minutes, and the precipitate was collected by centrifugation. The precipitate and centrifuge tube were washed carefully four times with 95 per cent ethanol to remove the alcohol- soluble 2,4-dinitrophenylhydrazone of pyruvate. The samples were then suspended in 0.4 ml. of 95 per cent ethanol saturated with sucrose (the sucrose served to prevent powdering of the dried precipitate) and transferred to a planchet with the aid of small volumes of ethanol. The precipitate was then dried under an infra-red lamp and counted. With the single precipitation procedure as described above phosphopyruvate precipitates were readily obtained which contained less than 0.2 per cent of the pyruvate-2-C14 present. By reprecipitation in the presence of additional unlabeled pyruvate, contamination by the pyruvate-2-G4 can be reduced to an exceptionally low value.

Isotopic Analyses for 018---For measurement of the 018 content, the oxygen of the samples was converted to or equilibrated with carbon dioxide and mass 46/44 ratios were determined with a mass spectrometer.3

Inorganic orthophosphate was converted to KHzPOh and heated to drive off KS0 essentially as described by Cohn (8). The O’* content of water samples was determined by either of two procedures. For one procedure an intimate mixture of 3.76 gm. of NaHC03, 0.47 gm. of NanSOs, and 0.19 gm. of NaHS03 was prepared and dried under vacuum over PZ05. A water sample of 20 to 50 mg. in a small stoppered vessel was incubated for 3 hours or more at room temperature with 4.4 mg. of the sulfite-bicar- bonate mixture per 50 mg. of water. The sample was then frozen, about 10 mg. of anhydrous NaHSO* were quickly added, and the tube was evacuated thoroughly while the contents were still frozen. The contents were then thawed to allow the acidic bisulfate to release CO2.4 The tube was again immersed in a dry ice-acetone mixture, and the CO2 was collected for mass spectrometry. Sulfite was used in this procedure because it markedly accelerates the reaction CO, + H20 ti H&OS (15) which limits the exchange of water oxygen with that of COZ; sulfite and sulfate oxygens do not exchange with oxygen of water under the conditions used (16).

3 Mass spectrometer analyses were made through the cooperation of Professor A. 0. C. Nier and Mr. B. L. Donnally, Jr., of the Department of Physics.

4 Citric acid is preferable to NaHS04 as an acidifying agent; with NaHSOa con-

siderable liberation of SOI may occur. The SO? does not, however, interfere in t,he mass 46/44 ratio determinations.

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The second procedure was a microadaptation (17) of the method described by Dostrovsky and Klein (18). Water from as little as 25 pmoles of KHzPOI was collected directly in an appropriate vessel containing 0.2 to 0.4 ml. of CO2 (standard temperature and pressure) and exposed to a hot Pt wire. Exposure for 15 to 20 minutes to a wire heated to a bright yellow color was necessary for complete equilibration. The water was then frozen out and the COZ was collected for mass spectrometry.

The 3-phosphoglycerate was isolated from reaction mixtures by barium precipitation following methods as given by Umbreit et al. (19) and by Neuberg and Lustig (20). The amorphous barium precipitates obtained in some experiments contained hexose diphosphate and inorganic phosphate as impurities; fortunately, complete removal of these was unneces- sary because they did not interfere in the decarboxylation procedure. The small sample sizes made more extensive purification undesirable.

The barium salt was converted to the silver salt essentially as described by Baer and Fischer (21) for preparation of silver phosphopyruvate. The silver salt darkened readily if exposed to light. The silver salt prepared from pure barium 3-phosphoglycerate contained the expected 3 atoms of silver per mole.

For determination of the amount of 018 in the carboxyl group of 3-phosphoglycerate 30 to 40 mg. of the silver salt of the acid were decarboxylated by treatment with Brz in CC& (22) ; this reaction has also been used by Little and Bloch for decarboxylation of acetic acid (23). The decarboxylation was carried out in an evacuated system followed by condensation of the Brz and CC14 in a dry ice-acetone bath (17). The COz formed was transferred to a suitable sample bulb with the aid of a Tijpler pump and used directly for mass spectrometer analysis. In Experiments 1 and 2 with rabbit muscle extract (Table I), the CO2 from the silver salt was collected as BaC03 by passage into Ba(OH)2. The BaC03 formed was treated under suitable conditions with concentrated HzS04 to liberate carbon dioxide. This procedure has the disadvantage that slight exchange of oxygen of the carbon dioxide with that of the water of the Ba(OH)* solution occurs.

The amount of Ols in the -O- oxygens of the phosphate groups of ATP, phosphocreatine, and 3-phosphoglycerate was determined by appropriate hydrolysis to form Pi and the Pi was isolated as MgNH4P04. The MgNH,PO* was freed of Mg* with a cation exchange column, then neutralized to pH 4.4 to 4.6 with KOH, and KHtP04 was precipitated by addition of alcohol.

To determine the O’* content of the phosphate of glycerophosphates, the glycerophosphates were hydrolyzed with alkaline phosphatasc by a procedure similar to that described for hydrolysis of 3-phosphoglycerate, except

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that the Pi formed was not precipitated until the end of the incubation period.

Miscellaneous Determinations-Inorganic phosphate was determined essentially as described by Sumner (24) and fructose-l ,6-diphosphate by the Roe reaction (25). Phosphocreatine phosphate and labile phosphate of ATP were determined by conventional procedures (19).

Results

Incorporation of Phosphate Oxygen into S-Phosphoglycerale-Considera- tions of probable enzymic reaction mechanisms suggested that the oxygen required for formation of 3-phosphoglycerate might be derived from inorganic phosphate. To test this possibility, experiments were conducted with rabbit muscle and yeast extracts in which fructose-l ,6-diphosphate was cleaved to triose phosphates and the glyceraldehyde-3-phosphate was oxidized with formation of 1,3-diphosphoglycerate in the presence of inorganic phosphate containing O18. Phosphate was transferred to adenosine diphosphate (ADP) by 3-phosphoglycerate kinase present in the extracts and the phosphoglycerate formed was isolated and decarboxylated. The mass 46/44 ratio of the carbon dioxide formed was determined as described under “Materials and methods.” The experimental conditions and results are given in Table I.

The results clearly demonstrate that oxygen from inorganic phosphate was present in the carboxyl group of the 3-phosphoglycerate. The amounts of excess 0’8 present in the carboxyl of 3-phosphoglycerate in Experiments 1 and 2 with rabbit muscle were 82.0 and 82.5 per cent of the total possible based on the initial excess 018 of the inorganic phosphate and on incorporation of 1 oxygen from phosphate into the carboxyl. The lack of the quantitative incorporation probably resulted principally from slight exchange of the oxygen of COZ with water in the isolation procedure used for Experiments 1 and 2 (see “Materials and methods”), as well as from depletion of the excess 018 of the inorganic phosphate during the reaction period. With the yeast preparation (Experiment 3) the amount of 0’8 in the carboxyl group was 34 per cent of the total possible based on the initial excess O1g of the inorganic ph0sphat.e. This apparently low yield was most likely the result of a relatively rapid exchange of inorganic phosphate oxygen with that of water which was catalyzed by the yeast preparation. The results with the muscle and the yeast preparations, together with the very probable high specificity of the enzyme reactions involved towards bonds cleaved, warrant the conclusion that 1 oxygen of t,he 3-phosphoglycerate carboxyl is derived from phosphate.

Transfer of Labeled Phosphate from S-Phosphoglycerate to Creatine--In experiments reported previously (26), transfer of phosphate from phospho-

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pyruvate to ADP by pyruvate kmase in HzO’* was shown to occur without incorporation of 018 into the labile phosphate groups of the ATP formed. These results did not conclusively rule out the possibility that oxygen of the C--O-P bond of the phosphopyruvate may have appeared in the ATP (26). The incorporation of oxygen from substrates, water, or any other source into the phosphate group transferred in an enzymic reaction may be checked by labeling the -O- oxygens of the phosphate group

TABLE I

Enzymic Incorporation of Oxygen from Inorganic Phosphate into Carboxjyl

of S-Phosphoglycerate

The reaction mixture for Experiment 1 contained 1.4 mmoles of Na fructose- 1,6-diphosphate, 5.89 mmoles of inorganic phosphate containing 0.364 atom per cent excess Or* (5.04 mmoles of added potassium phosphate, 0.85 mmole from enzyme preparation), 4 mmoles of sodium pyruvate, 1 mmole of KF, 0.6 mmole of creatine, and 15 ml. of a rabbit muscle extract in a total volume of 43.0 ml. at pH 7.2. The mixture was incubated for 4 hours at 37”, and then 5 ml. of 30 per cent trichloroacetic acid were added. Conditions for Experiment 2 same as for Experiment 1 except that 6.00 mmoles of phosphate containing 0.365 atom per cent excess 018 (5.15 mmoles of potassium phosphate, 0.85 mmole from enzyme preparation) and 1.0 mmole of fructose-1,6-diphosphate were used and the sample was incubated for 1; hours. The total volume was 39.1 ml. For Experiment 3 the reaction mixture contained 1.96 mmoles of fructose diphosphate, 9.8 mmoles of potassium phosphate (0.425 atom per cent excess 01*), 4.9 mmoles of NaHC03, 10.8 mmoles of acetaldehyde (added in five portions during incubation), 0.74 mmole of KF, and 11 gm. of Fleischmann’s dried yeast mixed with 20 ml. of Hz0 and 7.4 ml. of CC14 in a total volume of 78.8 ml. at pH 6.3. The incubation was for 9 hours at 37.5”.

Experiment No. Fructoseu~e~hosphate Atom per cent excess 0’8 in carboxyl group

1 Rabbit muscle 1.26 0.150 2 “ I‘ 0.95 0.151

3 Yeast 1.33 0.064

with O1*. If no loss of 01* accompanies the transfer reaction, the reaction must occur without the exchange of the -O- phosphate oxygens and with cleavage of the O-P bond between the phosphorus and the rest of the donor compound.

The lack of exchange of the -O- oxygens of phosphate in several enzymic phosphate transfers was demonstrated by use of 3-phosphoglycerate labeled with O’* in the -O- oxygens of the phosphate. In the presence of a suitable muscle extract the phosphate was transferred to creatine; thus the experiment provided a check on possible oxygen exchange reactions accompanying the phosphate transfers of the phosphoglyceromutase, py-

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ruvate kinase, and creatine kinase reactions, as well as the enolase reaction. In addition, myokinase and 3-phosphoglycerate kinase were probably present and active in the extract. Phosphocreatine formed was isolated by adding to the reaction mixture 3 ml. of 10 per cent CaClz in saturated Ca(OH)z solution at pH 8.8 to precipitate the inorganic phosphate. The phosphocreatine in the supernatant solution was hydrolyzed by acidifica- tion to the equivalent of 1 M HCl and heating at 100” for 7 minutes, and

TABLE II

Transfer of 018-Labeled Phosphate from S-Phosphoglycerate to Creatine

The reaction mixture for Experiment 1 contained 0.2 mmole of tris(hydroxy- methyl)aminomethane, 1 mmole of KCl, 0.1 mmole of MgCL, 0.010 mmole of ATP,

0.2 mmole of creatine, 0.101 mmoie of potassium 3-phosphoglycerate labeled with 0’8, and 19 mg. of an acetone powder of rat muscle in a total volume of 11.4 ml. at pH 7.2. After incubation for 40 minutes at 37.5”, 3 ml. of 10 per cent CaClz in a

saturated Ca(OH)z solution, pH 8.8, were added and the solution was adjusted to pH 8.8 with KOH to precipitate the inorganic phosphate, ATP, and ADP. The amounts of reactants were similar for Experiment 2, except that ATP was replaced

by adenylic acid, the amount of potassium 3-phosphoglycerate was 0.09 mmole, the total volume was 8.4 ml., and a dialyzed extract equivalent to 30 mg. of the acetone powder was used. After 40 minutes incubation, another equal portion of the enzyme

extract was added, the sample was incubated an additional 40 minutes, and then the CaClz solution was added.

Experiment No.

Atom per cent excess 0’8 of -O- phosphate oxygens

3-Phospho- glycerate Phosphocreatine

pmoles

1 76 2.18 0.244 0.234*

2 43 1.34 0.147 0.140

* This value is corrected for dilution by the ATP added and the ATP and phosphocreatine phosphate in the muscle extract; these corrections were not necessary

for Experiment 2.

the 018 content of the Pi formed was determined. Presence of ATP in the supernatant liquid from calcium precipitation would not interfere because both ATP and phosphocreatine would be expected to give Pi of the same Ols content.

The results given in Table II show that, within experimental error, the -O- oxygens of the phosphate in phosphocreatine had the same atom per cent excess Ols as those of 3-phosphoglycerate. Thus the enzymic reactions involved occurred without exchange of the -O- oxygens of the phosphate group transferred. If 1 oxygen had exchanged per group transferred, the expected atom per cent excess 018 in the -O- oxygens of

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phosphocreatine phosphate would have been 0.163 and 0.098 per cent for Experiments 1 and 2, respectively; exchange of 1 atom of oxygen per phosphate was thus readily detectable experimentally.

Transfer of Phosphate from ATP to Acetate in Hz018-As a test with an additional phosphate transfer reaction, measurements were made with a bacterial acetokinase preparation. Phosphate was transferred from ATP to acetate in Hz018 with hydroxylamine present to react, with the acetyl

TABLE III

Transfer of Phosphate from ATP to Acetate in Hz018

For Experiments 1 and 2, 8 mmoles of potassium acetate, 0.5 mmole of tris(hy-

droxymethyl)aminomethane, 0.1 mmole of MgS04,0.1 mmole of potassium ATP, and 7 mmoles of hydroxylamine hydrochloride were dissolved in Hz0’8, then neutralized to pH 7.4 with KOH, and the volume was made up to 10 ml. with HzO’* to give a

solution containing 1.27 atom per cent excess 01* in the HzO. A 0.1 ml. aliquot of an acetokinase preparation was added, the sample was incubated for 15 minutes at 29”, and 10 ml. of 10 per cent trichloroacetic acid were added. The inor-

ganic phosphate formed in Experiment 1 (from reaction of the hydroxylamine with acetyl phosphate) was isolated from the supernatant liquid as MgNH4POn. In Experiment 2 the ADP, together with residual ATP, was isolated by barium precipitation at pH 8.3, and the product obtained was hydrolyzed in 1 M HCl at 100” to

give inorganic phosphate.

I I I Atom per cent excess 0’8

ExpgAyt Source of inorganic phosphate ; Phosphate 1 transferred ~ Mas%%?atio ~+yc~df~

1 Acetyl phosphate

2 ADPt.

* The mass 46/44 ratio obtained with control non-labeled phosphate was 0.00419. t Expected value if 1 atom of oxygen from water appeared in the inorganic phos-

phate formed, with consideration of the dilution by carrier phosphate. $ The ADP fraction isolated contained approximately 13 per cent residual ATP.

phosphate formed, thus giving inorganic phosphate and acethydroxamic acid as final reactio; products. The 018 contents of the inorganic phosphate derived from the acetyl phosphate and of the easily hydrolyzable phosphate group of ADP were determined. The results given in Table III show that the presence of oxygen from water in the products was equivalent to much less than 1 oxygen per mole of phosphate transferred. These data warrant the conclusion that the phosphate transfer proceeded without exchange of the -O- oxygens of the phosphate group in a manner

analogous to those previously studied. Acid-Catalyzed Phosphate Transfer in HzO18-The interconversion of CX-

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and P-glycerophosphates resulting from heating in strong acid has been regarded as occurring by intermediate formation of a cyclic diester (27). To find out whether this non-enzymic phosphate transfer proceeded in a manner analogous to the enzymic transfer of phosphate, &glycerophosphate was heated with strong acid in the presence of HzO’*. The experimental conditions and results are given in Table IV. The acidity and heating time exceeded considerably the use of 3.5 N sulfuric acid and 3 hour reflux reported by Chargaff as adequate for conversion of /3- into cr-glycerophosphoric acid (27). The results show that marked exchange of the phosphate oxygens with those of water had occurred in the refluxed sample. The much smaller exchange noted in the control sample may

TABLE IV

Phosphate Oxygen Exchange during Intramolecular Transfer of Phosphate Catalyzed by Acid

Two solutions containing 7.35 mmoles of fi-glycerophosphoric acid in 9 ml. of 4 N HCI with 0.894 atom per cent excess Or8 in the water were prepared. One solution (control) was allowed to stand at room temperature; the other was refluxed for 3 hours. A second refluxed sample contained inorganic orthophosphoric acid in place of the p-glycerophosphoric acid. Inorganic phosphate formed by slight hydrolysis in the refluxed sample of fl-glycerophosphoric acid was removed as MgNHdPOd, and

the 0’8 content of the -O- oxygens of the phosphate of glycerophosphate was determined by alkaline phosphatase hydrolysis as described in the text.

Source of phosphate analyzed Mass 46144 ratio of CO2 Atom per cent excess 0’8 in phosphate

Control fl-glycerophosphate. ............... Refluxed “ ................

‘I orthophosphate ..................

0.00568 0.08 0.0131 0.44

0.00429 <0.03

have resulted from the known catalysis by alkaline phosphatase of exchange of Pi oxygen with oxygen of water; in the experiments the Pi formed by alkaline phosphatasc hydrolysis TV-as not precipitated as MgNH4P04 until the end of the incubation period. The data give strong evidence that the phosphate transfer reaction was accompanied by exchange of phosphate oxygen with that of water.

lkzymic Incorporation of Pyruvate-b-C14 into Phosphopyruvate-Experi- ments were conducted to measure the dependence upon various reaction components of the incorporation of pyruvate-2-Cl4 into phosphopyruvate as catalyzed by pyruvate kinase. The data in Table V show the effect of omission of enzyme, ATP and ADP, phosphopyruvate, or K+ from the complete reaction system. The marked acceleration of the exchange by the addition of ATP and ADP is apparent. The time-course of the dependence of the exchange on the addition of ATP and ADP has been

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given in an earlier incomplete report (26). The acceleration of the exchange by addition of phosphopyruvate shows that the radioactivity found in the barium fraction containing phosphopyruvate in the complete system was not the result of net formation of phosphopyruvate from ATP. The effect of added K+ rules out the possibility that the transfer reaction occurred in two steps, the first of which did not require K+ and involved transfer of phosphate to an intermediate. The small fraction of the radioactivity of the added pyruvate incorporated into the phosphopyruvate is a reflection of the relatively slow rate of the reaction at equilibrium as con-

TABLE V

Incorporation of Pyruvate-l-C14 into Phosphopyruvate Catalyzed by Pyruvate Kinase

The complete reaction mixture contained in 1.0 ml. total volume, pH 7.4, 0.02 M

sodium phosphate buffer, 0.15 M KCI, 0.005 M MgSOa, 0.001 M of a mixture of sodium ATP and ADP (approximately 10 per cent of ADP), 0.001 M sodium pyruvate-2-W

equivalent to approximately 33,000 c.p.m., 0.002 M sodium phosphopyruvate, and rabbit muscle pyruvate kinase. Incubation was for 3 hours at 37”, following which the phosphopyruvate was isolated and its radioactivity determined as described in the text. The pyruvate kinase at l/100 the concentration used catalyzed the trans-

fer of 0.19 pmole of phosphate from phosphopyruvate to ADP per ml. of reaction mixture in a similar medium without added pyruvate but with added hexokinase and glucose.

Reaction component omitted

--

Enzyme .............................................. ATP-ADP ........................................... Phosphopyruvate. ....................................

Potassium ............................................ None ................................................

Observed radioactivity of phosphopyruvate fraction

c.p.m.

58 63

115

112 635

trasted to the rate when phosphopyruvate reacts with ADP in the absence of ATP and pyruvate.

DISCUSSION

The demonstration that an oxygen from phosphate appears in the car- boxy1 group of 3-phosphoglycerate formed enzymically from glyceraldehyde-3-phosphate shows that the transfer of phosphate from 1,3-diphosphoglycerate to ADP takes place with the cleavage of the O-P bond of the C-O-P linkage. The incorporation of phosphate oxygen into the C-O-P linkage of 1,3-diphosphoglycerate probably results from a nucleophilic displacement by phosphate oxygen on the acyl carbon of a 3-phosphoglyceryl-S-enzyme intermediate formed in the glyceraldehyde-3- phosphate dehydrogenase reaction (28). Such a mechanism is in harmony

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with the results of Cohn showing that glyceraldehyde-3-phosphate dehydrogenase does not catalyze exchange of phosphate oxygens with water (9).

The results reported herein also demonstrate that phosphate transfer in the phosphoglyceromutase, pyruvate kinase, and creatine kinase reactions occurs without exchange of the -O- oxygens of the phosphate group transferred with oxygen from any source. Further, they show, that, in the acetokinase reaction, oxygen of phosphate transferred from ATP or of the ADP formed has not exchanged with that of mater. Similar results have been obtained with the 3-phosphoglycerate kinase (9) and hexokinase and myokinase reactions.5 Thus .in these enzymic phosphate transfers the -O- oxygens of the phosphate donor remain as such in the phosphate acceptor, and the transfer reactions occur with cleavage of the bond between the P of the phosphate and an 0 or N of the donor molecule. The phosphate transfer reactions may be regarded as resulting from nucleophilic attack of an 0 or N of the acceptor molecule on the P atom of the donor (26, 29).

Biicher has presented evidence that the acyl phosphate of 1,3-diphosphoglycerate is bound to the same position on 3-phosphoglycerate kinase as the terminal phosphate of ATP (30) ; this is in logical agreement with the mechanism suggested by the studies with O1*. The phosphate group to be transferred from either phosphate donor may be regarded as being bound at the same site on the enzyme and rendered labile to nucleophilic attack by the acceptor. Additional kinetic studies are necessary to estab- lish whether the concept of single binding sites for the phosphate group transferred is valid for various phosphate-transferring enzymes.

A probable function of Mg* in the catalysis is to combine with the phosphate group to be transferred, thus increasing the residual positive charge on the phosphorus atom and its susceptibility to nucleophilic attack (26). The phosphate group of donor substrates such as phosphopyruvate and phosphocreatine would be expected to have weak affinity for Mg* com- parable to that of glucose-l-phosphate (31) and glycerophosphate (32) ; the enzyme may facilitate combination of the phosphate group to be transferred with Mg++. Present evidence is insufficient to warrant speculation about the manner of such combination.

Although the simplest explanation of the results of the O’* studies is the postulate of a direct transfer of phosphate from donor to acceptor, the occurrence of the reaction without exchange of -O- oxygens of the phosphate does not rule out the possibility of formation of enzyme-phosphate intermediates. Formation of such intermediates by the same mechanism

5 See discussion by M. Cohn in McElroy, W. D., and Glass, B., The mechanism of enzyme action, Baltimore, p. 520 (1954).

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would not lead to exchange of the -O- oxygens. The studies of the pyruvate kinase reaction with pyruvate-2-Cl4 (Table V and Boyer and Harrison (26)) and of the acetokinase reaction with P32 and Cl4 (33) give strong evidence against the formation of enzyme-phosphate intermediates in these enzymic reactions. In contrast, formation of an enzyme-phosphate intermediate in the phosphoglucomutase reaction appears to be well established (7). The phosphate in a-glucose-l-phosphate is on the oppo- site side of the glucopyranose ring from the phosphate in glucose-6-phosphate; this makes unlikely the direct transfer of phosphate from the 1 to the 6 position by nucleophilic attack of the acceptor oxygen on the phosphorus atom (34). The intermediate formation of an enzyme-phosphate would thus appear logical in the phosphoglucomutase and in other intramolecular phosphate transfers for which similar considerations apply.

Phosphate transfer by phosphatases (35, 36) would be expected to con- form to the mechanism suggested above; phosphatases have been shown to catalyze cleavage of O-P bonds and hydrolysis may be regarded as a transfer of the phosphate group to an oxygen of water (29).

The stability of phosphate oxygens in enzymatic phosphate transfer reactions suggests that explanation of the rapid exchange of phosphate oxygen catalyzed by liver mitochondria (9) is to be sought in reactions in which inorganic phosphate participates. Other studies in this laboratory have shown that the uptake of inorganic phosphate accompanying elec- tron transport is readily reversible in liver mitochondria, and the reversal of the initial reactions leading to the uptake of the inorganic phosphate has been suggested as an explanation for the rapid exchange of phosphate oxygen (37).

SUMMARY

Experiments with Ols have shown that oxygen from inorganic phosphate appears in the carboxyl group of 3-phosphoglycerate formed by enzymic oxidation of 3-phosphoglyceraldehyde and transfer of phosphate from 1,3- diphosphoglycerate to ADP. The phosphate transfer reaction thus takes place with cleavage of the O-P bond in the C-O-P linkage of 1,3-diphosphoglycerate. Incorporation of phosphate oxygen into the C-O-P linkage suggests that an acyl enzyme formed in the glyceraldehyde-3- phosphate dehydrogenase reaction is cleaved by nucleophilic attack of phosphate oxygen on the acyl carbon.

The enzymic reactions of muscle extract by whirh the phosphate of 3- phosphoglycerate is transferred to creatine to form phosphocreatine occur without exchange of excess O** originally present in the three -O- oxygens of 3-phosphoglycerate. The enzymic transfer of phosphate from ATP to acetate by a bacterial enzyme preparation occurred without incor-

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poration of 018 from HO’*H into the ADP or acetyl phosphate formed. These results show conclusively that, in the reactions catalyzed by phosphoglyceromutase, pyruvate kinase, creatine kinase, and acetokinase, the three -O- oxygens of the phosphate donor remain as such in the phosphate acceptor and that the transfer reactions occur with cleavage of the bond between the P of phosphate and an 0 or N of the donor molecule. Also the data demonstrate that the enolase reaction proceeds without any exchange of phosphate oxygen.

The enzymic phosphate transfer reactions studied and other similar phosphate transfer reactions are regarded as proceeding by nucleophilic attack of an 0 or N atom in the acceptor molecule on the P atom of the donor phosphate compound. The results do not rule out possible formation of enzyme-phosphate intermediates. Additional implications of the data are discussed.

In contrast to the enzymic transfer, treatment of P-glycerophosphate with acid under conditions which cause intramolecular migration of the phosphate group results in incorporation of 018 from HO18H into the phosphate group. This gives support to the diester mechanism proposed for such phosphate transfer.

The incorporation of pyruvate-2-CF4 into phosphopyruvate in the presence of pyruvate kinase is dependent upon the presence of K+ as well as ADP. This is evidence against the formation of an enzyme-phosphate intermediate in the pyruvate kinase reaction.

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W. H. Harrison, P. D. Boyer and A. B. FalconePHOSPHATE TRANSFER REACTIONS

THE MECHANISM OF ENZYMIC

1955, 215:303-317.J. Biol. Chem.

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