SYNTHETIC ACTION OF PHOSPHATASE”

I. EQUILIBRIA OF BIOLOGICAL ESTERS

BY OTTO MEYERHOF AND HARRY GREEN

(From the Department of Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia)

(Received for publication, December 1, 1948)

While several attempts have been made to calculate the free energy con- tent of the energy-rich phosphate bonds (l-4) in intermediary carbohydrate metabolism, comparatively little is known about the energy value of the bonds of ordinary phosphate esters. We have undertaken to bridge this gap by measuring the enzymatic equilibria reached in the synthesis of various biological phosphate esters. As alcohols for phosphorylation we have used the common hexoses (glucose, fructose, mannose, and galactose), glycerol, and glyceric acid. The results with glyceric acid will be reported separately.

Although the synthetic action of unpurified phosphatase from various organs was observed at an early date with polyhydric alcohols and sugars (5-7), the enzymatic equilibrium has been measured with glycerol (7, 8) and methyl alcohol (9) only.

We have obtained the synthesis of the phosphate esters of all the natural sugars studied in the presence of alkaline phosphatase. Glucose and fructose were also phosphorylated in the presence of acid phosphatase de- rived from the (human) prostate gland.

Although these phosphatases seem unspecific regarding the nature of the sugar or polyhydric alcohol, t,he synthesized esters were all of bio- logical configuration; i.e., the hexose was phosphorylated in the 1 or 6 position or in both. Glycerol and glyceric acid were phosphorylated pre- ponderantly on the primary alcoholic group with both alkaline and acid phosphatase.

K, the equilibrium const.ant, is calculated from the concentrations at equilibrium according to the equation

KJ @; inor anic phosphate1 X [alcohol] [water] X [ester] (1)

*This work was aided by grants from the American Cancer Society, recom- mended by the Committee on Growth of the National Research Council, and the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service.

655

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656 SYNTHETIC ACTION OF PHOSPHATASE. I

in which the brackets refer to molar concentration. In determining the standard free energy (AFO) from the equation

AF” = -RT In KQ (2)

the convention proposed by Lewis (10) has been followed, which assumes the standard state of the solutes to be 1 molal, but that of water to be pure water and equal to 55.5 molal.’ Therefore,

K" = K x 55.5

and AF” = -tZ2 In (K X 55.6) (3)

The end-points of synthesis reached for the given time intervals repre- sent the true thermodynamic equilibria, at least for t,he main ester com- ponent formed. This presupposes, of course, that the activity of the enzyme is high enough to reach such an end-point. When this is doubtful, the experiment must be discarded. In cases in which several component esters are formed the equilibrium must be calculated for each single com- pound. The accuracy of the experiments, however, permitted an exact calculation for the main component only and an estimate for the other components present.

Materials

Phosphatase-Alkaline intestinal phosphatase purified according to Schmidt and Thannhauser (11) and a sample of acid phosphatase from human prostate gland prepared by Dr. Schmidt2 were used.

Xugars-The sugars used were of the purest commercial preparations available: Merck anhydrous glucose, Merck levulose, Paragon galactose, and Fisher Scientific mannose.

Glycerol-Baker’s Analyzed glycerol containing 4.3 per cent moisture by weight was used.

Methods

All incubations were conducted at 38”. Besides measuring the total amount of esterified phosphate after remov-

ing the inorganic phosphate by magnesia mixture, we have in every case

1 We thank Dr. H. Borsook for a valuable discussion of these points. The nu- merical value of K in this ease is the same whether molar or molal concentrations are used.

2 We thank Dr. Gerhard Schmidt who has kindly supplied us with several batches of alkaline and acid phosphatase prepared by himself, and also privately informed us of some new modifications in the procedure of Schmidt and Thannhauser which we have adopted to increase considerably the total yield of the purified enzyme.

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0. MEYERHOF AND H. GREEN 657

isolated the barium salts and analyzed them according to the usual pro- cedures.

Phosphate-Phosphate was determined by the method of Fiske and Subbarow (12) as modified by Lohmann and Jendrassik (13).

Ketose-The ketose content was determined by the method of Roe (14). Hydrolysis-The hydrolysis curves of the barium-free esters were de-

termined according to the procedure of Lohmann (15) at 100“ and in N HCl.

Hexose Diphosphate-The amount of fructose-l ,6-diphosphate present was determined with muscle zymohexase in the presence of KCN accord- ing to the procedure of Meyerhof, Ohlmeyer, and Miihle (1) and Herbert et al. (16).

(Y- and fl-Glycerophosphate-For determining the amount of a-glycero- phosphate in the presence of the p isomer the periodate method of Fleury and Paris (17) was used.

The initial composition of the concentrated sugar solutions was found by measuring the specific gravity, the optical rotation, and the content of inorganic phosphate. In the course of the experiments it was ob- served that a slow enolization took place in the sugar solutions incubated at 38” independently of any action of the enzyme, resulting in the forma- tion of the corresponding isomers. In each experiment, therefore, the extent of the enolization was determined, i.e., the aldose content in the fructose experiments and the ketose content in the aldose experiments, by measuring the increase or decrease in the Roe value of free ketose. For calculation of the equilibrium constants, these values were deducted from the concentration of the reacting sugars. Similarly, the concentra- tions of the corresponding phosphate esters formed therefrom were de- ducted from the total phosphate esters in the respective equilibria. Since the percentage of the enolized free sugars was roughly, and, in some cases, exactly, equal to the percentage of the corresponding phosphate ester, the K values were not appreciably affected.

Experimental

A representative experiment with fructose is described in detail as il- lustrative of the general procedure and results.

Fructose-A solution of fructose (2.7 gm.), 0.9 ml. of 2.75 M Na2HP04, and 1.2 ml. of purified intestinal phosphatase (activity, 3774 units per ml. as defined by Schmidt and Thannhauser) was incubated at 38”. The concentration of organic phosphate was determined periodically. After 72 hours of incubation, when the slope of the curve obtained by plotting the organic phosphate concentration against time in hours (Fig. 1) in- dicated the attainment of equilibrium, the remaining reaction mixture

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658 SYNTHETIC ACTION OF PHOSPHATASE. I

was deproteinized with 40 per cent trichloroacetic acid. After removal of the inorganic phosphate with magnesia mixture, the barium salts were precipitated with barium acetate and alcohol, purified by reprecipitation, and dried. Weight of barium salts, 95 mg.

Analysis-22 mg. Ba salts in 14.0 ml. solution (Ba++ removed) Organic P . . . . . . . 62.7 y P per ml., 4.00% Inorganic P.. . . . . . . . . 0.0 “ “ ‘( “ 0.00% Fructose-l, 6-diphosphate . 2.8 “ ‘6 “ “ 4.5y0 of organic P or

2.25% of hexose content

FIG. 1. Speed of synthesis. Phosphorylation of fructose (FR), mannose (MA), and glycerol (GLY), at 38”, pH 8.5, in the presence of purified alkaline intestinal phosphatase and NazHPOd. Ordinate, molar concentration X lo* of phosphate es- ter formed. Abscissa, time of incubation in hours.

The hydrolysis values and the corresponding literature values for the component esters are given in Table I. For each time period of hydroly- sis an equation of the form ax + by + cz + dw = A may be written, where a, b, c, and d represent the literature values corresponding to the com- ponent esters, CC, y, x, and w. A is the amount of inorganic P liberated. The solution of these equations by the method of simultaneous equations or of least squares gave the following quantitative distribution of esters: fructose-l-phosphate, 64.3 per cent; fructose-6-phosphate, 29.4 per cent; fructose-l ,6-diphosphate, 2.3 per cent; glucoseB-phosphate,3 4.1 per cent.

3 The iodometric method of Willstatter and Schudel (21) as modified by Robison and MacLeod (22) was found inapplicable for the determination of the small amount of glucose-6-phosphate in the above mixture.

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0. MEYERHOF AND H. GREEN 659

The main result of the fructose experiments (Tables II and III) may be briefly stated as follows: enzymatically only those esters are formed which are derived from the sugar without change in configuration. The aldose- 6-phosphate may be accounted for by the phosphorylation of the aldose

TABLE I

Distribution of Esters in Fructose Experiments

Literature values, hydrolysis Hydrolysis at 100” in 1 N HCl

Time I P per ml. 1 liberated Hydrolysis

Fructose-l- ! Fructose-6- phosphate phosphate

(18) (19)

per c&i1 +er cent 70.2 9.6 92.4 13.0

100.0 24.4 100.0 83.6

I

-I-

‘ructose-1,6- diph~~o~bate

Glucose-6- phosphate

(1% ./. min.

7 15 30

180

Y

31.6 38.8 45.4 57.2

per cent

50.4 61.9 72.4 91.2

-

TABLE II

Formation of Fructose Phosphates

In all experiments but the last the pH was 8.5; in Experiment VII the pH was 5.8. The enzyme activity in all experiments but No. IVA was about 1000 units per ml.; in Experiment IVA it was 3770 units per ml. The initial water concentration in every experiment was 30.0 M.

Initial molar concentration Final molar concentration

I / , - --.-I I

FlXC- tose

Pbos- Total pbate Fructose ester

x 102

FNC- tose-l- phos- phate x 10’

FlUC- tose-6- phos- phate x 101

FNC- tose-1, Gbl- En01 K 6-di- case-6- global’

pbos- pbos-

pxh$ pbate x 102

4.71 4.02 4.02 4.02

4.02

Experi- ment No

--

M IV IVA V

Time

hrs.

240 268

72 552

168

- -__ ~-- - per cent

1.55 0.157 0.00 11 1.24 0.144 0.262 3 1.17 0.241 0.458 11.9 1.34

-- --

0.115 0.170 12 3.44

-..-- -__ ~

0.61 (4.65)t 6.0 0.675 3.52 6.50 0.629 3.84 6.38 0.655 3.48 5.88

_____--

0.700, 3.51 2.6

4.22 4.10 3.82

2.10 1.88 1.34

0.91

._

- VII 1.41

* Based upon total organic esters at equilibrium without correcting for enoliza- tion.

i Value not corrected for enolization.

resulting from the partial enolization of the fructose. The high con- centration of fructose-l-phosphate (65 per cent) compared to that of fructose-6-phosphate (28 per cent) indicates that the phosphate bond in the 1 position possesses much less energy than in the 6 position. More- over, fructose-l-phosphate facilitates the phosphorylation of the 6 posi-

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660 SYNTHETIC ACTION OF PHOSPHATASE. I

tion to give 2.3 to 4.17 per cent fructose-1,6-diphosphate (and twice as much based on the phosphate esterified), as is evident from the following consideration: since the total ester represents only 1.5 per cent of the free fructose present, on the basis of random phosphorylation of the 6 position of fructose-l-phosphate (as would occur if free fructose were present in the same concentration) much less should form. The formation of about 10 times this amount of the diester shows that less energy is needed to esterify fructose-l-phosphate in the 6 position than is needed to esterify fructose itself in the same position, and even to esterify fructose in the favored 1 position. K for fructose-l-phosphate at pH 8.5 is 1.79; at pH 5.8, 5.5; K for fructose-6-phosphate at pH 8.5 is 4.2; at pH 5.8, 8.67. Finally, for fructose-l, 6-diphosphate starting with fructose-l-phosphate, K at pH

TABLE III K Values for Fructose Esters

Experiment No. PH

IV 8.5 IVA 8.5 V 8.5

Average.. . . . . 8.5

VII 5.8

Fructose-1,6-diphosphate

Global Fructose-l- Fructose-6- phosphate phosphate From FNC-

Direct tose-l-phos- phate

-

1.24 1.69 3.40 46.3 0.545 1.17 1.82 3.97 51.7 0.536 1.34 1.81 5.15 29.1 0.314

-..-.- I , I I I

1.25

I

1.77

I

4.2 42 0.465

- 3.44 5.58 8.67 69.1 0.275 -

8.5 is 0.465; at pH 5.8, 0.275; starting with fructose, K at pH 8.5 is 42; at pH 5.8, 69.

Glucose, Galactose, and Mannose-The experimental conditions and procedure were essentially similar to those employed in the fructose ex- periment described previously. The ketose phosphate was determined by the method of Roe (14) and calculated as fructoseS-phosphate according to Umbreit (23).

Analysis-10 mg. Ba salt of glucose phosphate per 5.0 ml. (Ba++ removed). Organic P content.. 90.5 y per ml. (0.525 mg. hexose) Roe value. . 23.5 (‘ Cc “ fructose

“ I‘ corrected for fructose-6-phosphate. 38.8 “ “ “ “ = 7.4yo of hexose content

The hydrolysis values and the corresponding literature values for the component esters are given in Table IV. By correlating these values as described previously, the following quantitative distribution of the esters

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0. MEYERHOF AND H. GREEN 661

was obtained: glucose-l-phosphate 9.0 per cent, glucose-6-phosphate 83.6 per cent, fructose-6-phosphate 7.4 per cent.

Since the equilibrium ratio of glucose-6-phosphate to glucose-l-phosphate is about 20 (25), not more than 5 per cent of the latter ester can form. Because of the uncertainty of the exact distribution of glucose-l-phosphate and glucose-g-phosphate, for the purpose of calculating the equilibrium constant the amount of the latter ester was taken as the difference between the hexose content of the ester as calculated from the organic phosphorus and the ketose content as determined by the method of Roe. The same reasoning applies to the galactose and mannose phosphates for which the equilibrium ratios of the 6- to l-esters are not known; the hydrolysis curves, however, indicate the presence of from 7 to 10 per cent of the l-ester.

TABLE IV

Distribution of Esters in Glucose Experiments Total organic P per ml. = 201 r; hydrolysis at 100“ in 1 N HCI.

Literature values, hydrolysis Tiie P per ml.

liberated hydrolysis Glucose-l- Glucose-6- Fructosed-

phosphate (24) phosphate (19) phosphate (19)

per ce?zt per cent per cent

0.4 9.6 100 1.0 13.0

1.5 24.4 5.2 59.6 9.7 83.6

min. 7 19.0

15 25.0 30 31.5 90 42.0

180 , 61.0

per cent 9.45

12.4 15.7 23.3 30.3

Tables V and VI summarize the results of the aldose experiments. All the ketose esters herein may be considered to arise by the partial enoliza- tion of the aldose sugar and subsequent phosphorylation. If this is taken into account, then practically only aldose-g-phosphate is formed from the corresponding aldose, besides a few per cent of the corresponding aldose- l-phosphate. The average value of K for glucose-6-phosphate at pH 8.5 is 2.02; at pH 5.8 K lies between 4 and 5. The one experiment (No. VII) with a dilute solution of glucose gave a K value of 1.4. For ga- lactose-6-phosphate at pH 8.5, K is 2.32. The one experiment with man- nose gave a somewhat smaller K, 1.40.

GZyceroZ-The procedure of sampling and of isolating the barium salts was essentially similar to that employed in the sugar experiments. The excess NH3 was removed, prior to the precipitation of the barium salts, by gentle boiling for a few minutes. The barium glycerophosphates were repeatedly purified by reprecipitation until the periodate determination of the CY isomer gave a constant value.

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662 SYNTHETIC ACTION OF PHOSPHATASE. I

An analysis of Experiment II follows: Twice purified barium salt; 10 mg. per 5.0 ml. (Ba++ removed); organic P = 150 y per ml., 0.445 mg. of glycerol. A 2.0 ml. sample required 2.23 ml. of 0.00850 N 1~ for titration

TABLE V

Formation of Glucose Phosphates

The pH in all experiments but the last was 8.5; in Experiment VI the pH was 5.8. The enzyme activity was 1000 units per ml. in all experiments.

Initial molar concentration il Final molar concentration K

Experi- ment No.

Ghl- Total case-6- ester phos- x 102 phate

x 102 __I_

2.36 (2.12: 3.24 4.53 4.20 5.5 4.71 2.16 2.04

-___

--

2.17

FIX- tose-6- En01 phos- phate x 102

__~ ger c‘m

0.24

0.335 4.6 0.781 5.5 0.082 3.8 --

--

GlU- Global cased- phos-

phate - __-

2.27 2.38 2.3 2.07 2.13 1.89 2.09 1.4 1.43

--

2.00 2.02 --

4.3

Glucose

A 3.93 0.405 C 3.93 0.527 IV 4.2 0.675 V 4.2 0.752 VII 2.38 0.555

--__ --

Average. 1 I I

hrs.

140 170 268 550 216

29.3 3.9 29.3 3.9 27.8 3.96 27.8 3.93 41.5 2.27

VI / 170 / 4.2 ( 0.655 / 27.8 4.18

Value in parentheses not corrected for enolization.

TABLE VI

Formation of Galactose and Mannose Phosphates

pH 8.5; enzyme activity, 1000 units per ml.; 9 per cent enolization.

K Initial molar concentration Final molar concentration

Experi- mentNo. Sugar

Aldose- 6-phos- phate

__-

2.30 2.33 1.40

Time Aldose- Ketose 6- hos-

Yl phas-

p ate phate x 102 x 104

--

2.78 0.29f 2.76 0.27C 5.71 0.528

Global

1.82 2.13 2.35 1.41

hrs.

170 528 168 264

2.60 0.76 37 2.57 2.92 2.78 0.987 37.2 2.50 3.29 2.78 0.987 37.2 2.50 3.01 4.2 0.653, 27.8 3.78 6.24

M Galactose V “

VII ‘( VI Mannose

of the excess NaXAsOa (0.01 N), equal to 0.438 mg. of glycerol per ml., 98 per cent cr-glycerophosphate.

The procedure was repeated with thrice purified barium salt; 10 mg. per 5.0 ml. (Ba++ removed); organic P = 164 y per ml., 0.487 mg. of glycerol. A 2.0 ml. sample required 2.36 ml. of 0.00875 N IZ for titration

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0. MEYERHOF AND H. GREEN 663

of the excess Na3As03 (0.01 N), equal to 0.475 mg. of glycerol per ml., 97.5 per cent cr-glycerophosphate.

The equilibrium for glycerophosphate has been quantitatively deter- mined by Kay (7) with crude alkaline intestinal phosphatase and by Ohlmeyer (8) with acid phosphatase from human prostate gland. The former found K = 0.68 at pH 8.5 and the latter K = 1.8 at pH 5.8. Our own values (Table VII) are quite similar: 0.63 for alkaline phosphatase (pH 8.5) and 1.5 for acid phosphatase (pH 5.8). In one essential point, however, our results differ from those of Ohlmeyer. According to him, equal amounts of the Q! and p ester were formed. We found, however, that with both alkaline and acid phosphatase 98 per cent of the glycero- phosphate formed consisted of the LY isomer. That this comparatively

TABLE VII

Formation of Glyeerophosphate The pH in all experiments but the last was 8.5; in Experiment 1 the pH was 5.8.

The enzyme activity in all experiments was about 1000 units per ml.

I I Initial molar concentration Experi-

merit No Time

~- hrs.

2 168 3 144 4a 700 4b 800

Average. . . . .

1 48

Glycerol Phosphate Hz0

7.10 0.405 1.7 0.480

11.19 0.0863 11.13 0.0863

0.408 26.9 7.02 6.1

-

-.

-.

27.00 48.8 10.3 10.3

I- Final molar concentration

Glycerol Total este x 102

I

6.99 11.0 1.677 2.26

11.14 5.52 11.13 5.83

_____

cz ester -- ger cent

98 99 87 84

99

_-

_-

-

K

Global OL --

0.69 0.71 0.695 0.70 0.61 0.69 0.52 0.61 --

0.63 0.68 ___-

1.48 1.49

high value was not due to the contamination of free glycerol resulting from insufficient separation was proved by adding glycerol to a mixture of 50 per cent (x and 50 per cent p isomers and following the same procedure for the isolation and purification of the Ba salt. The same distribution of the (II and p isomers was found before and after glycerol treatment. Of still greater importance was the transformation of the 50: 50 mixture in the presence of phosphatase into a mixture of 95 per cent (Y- and 5 per cent 8-glycerophosphate in 3 to 5 days.

The great preponderance of the a! isomer is not unexpected since there are two Q! positions for one p position; furthermore, the phosphate ester of the secondary alcohol can be assumed to have a higher energy content than that of the primary alcohol. This is best demonstrated by the analo- gous 3-phosphoglyceric and 2-phosphoglyceric acids which, at equi-

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664 SYNTHETIC ACTION OF PHOSPHATASE. I

librium, distribute in the ratio of about 4: 1 (26). The high periodate value precludes the possibility that any appreciable amount of the biochemically unknown glycerodiphosphate was formed.

Finally, it seems logical that the much lower value for the K of the primary alcoholic phosphate ester of glycerol compared with the corres- ponding esters of the hexoses is also mainly due to the presence of two equal primary alcoholic groups in every molecule. Indeed, the K value is

TABLE VIII Acceleration of Phosphorylation of Glycerol

1.7 M glycerol, 0.48 M phosphate, 48 M HnO, 0.0247 M organic phosphate added. Sample, 0.5 ml.

Phosphate compound added --

15 min. 30 min. 60 min. 120 min.

Control. ...................... Phosphocreatine. ............. Acceleration, ‘%. .............. Control ....................... 14.1 Phosphocreatine. ............. 42.0 Acceleration, yc ............... 200 Control ....................... 10.8 Phosphocreatine. ............. 41.0 Acceleration, %. .............. 272

16.2 43.0

165 20.1 74

270

19.8 64.0

220 34.2 97.0

185 34.2 94.0

175

32.1 77.7 91.0 221.0

184 185

Control, ...................... Fructose-l-phosphate .......... Acceleration, %. .............. Control ....................... Fructose-l-phosphate. ......... Acceleration, %. ..............

31.2 51.0 68

36.0 72.9 70.8 143.0 97 96 38.1 74.1 75.0 146.0 97.4 97.5

Control ....................... , 16.5 27.0 62.4 Glucose-l-phosphate. ......... / 36.0 62.0 144.0 Acceleration, ‘%. .............. I 118 130 131

Glycerophosphate formed, y P per 0.5 ml.

-

Equilih- rium

(w hrs.)

266 266

372 321

310 345

325 325

331 331

295 321

a little less than half that of the most favored components of the hexose esters.

Speed of Synthesis-Fig. 1 presents some rate curves for attainment of the equilibrium in the synthesis of hexose phosphate and of glycero- phosphate. A considerably longer time is required than would be ex- pected from the activity of the enzyme in the direction of hydrolysis and from the value of K. The latter constant, therefore, cannot be calculated from the simple formula,

k synthesis KS----- ktwazcw.

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0. MEYERHOF AND H. GREEN 665

A similar disparity was encountered by Biicher (27) in the study of other enzymatic phosphorylating reactions. Under our conditions the inorganic phosphate exerts a special affinity for the enzyme, thereby repressing the competitive affinity of the other reactants. The net effect is a retarda- tion of the reaction rate not anticipated from the law of mass action.

While the speed of synthesis, consequently, cannot be theoretically explained by the activity of the enzyme and the position of equilibrium,

GP(+PG) sm. c_____________________ ----------- /*-

c ,=-=.w

12345 HOURS 91

FIG. 2. Speed of enzymatic synthesis of glycerophosphate (GP) with and with- out added phosphocreatine (PC), at 38”, pH 8.5, in the presence of purified alkaline intestinal phosphatase and NasHPOd. v, synthesis of glycerophosphate (control); V, synthesis of glycerophosphate in presence of added phosphocreatine; 0, de- crease of phosphocreatine (control) ; l , decrease of phosphocreatine in presence of glycerol (G).

a very interesting phenomenon was observed during the enzymatic syn- thesis of glycerophosphate in the presence of organic phosphate com- pounds of higher energy content (Table VIII). Depending upon the energy content of the added organic phosphate, the speed of synthesis of glycerophosphate was increased 100 to 300 per cent (Fig. 2). At the same time the dephosphorylation of the added organic phosphate was delayed. Whether this phenomenon is related to the transphosphorylation be- tween aryl phosphates and certain alcohols observed by Axelrod (28) with acid citrus phosphatase is being investigated in our laboratory.

Method-The amount of synthesized glycerophosphate was deter-

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666 SYNTHETIC ACTION OF PHOSPHATASE. I

mined as the difference between the total organic phosphate, and that of the added organic phosphate. The latter, for each time interval, was determined by acid hydrolysis. A control incubation was run simul- taneously in order to follow the rate of hydrolysis of the added. organic phosphate in the absence of glycerol.

DISCUSSION

The calculation of the equilibrium constants of phosphorylation in this investigation has been based upon molar concentrations. A more exact calculation, however, involves the use of activities; e.g., by apply- ing the formulas of Lewis the activity coefficient of 2.38 M glucose would be 1.26. Since data are not available for all the substances and for all the concentrations, activity coefficients were not employed. Besides, the use of the latter would not make the figures in the foregoing more con- sistent, since the tendency of the K value to become larger with increasing concentration would be even more pronounced.

Although in those equilibria in which several esters are formed the de- termination of those present in smaller amounts is less accurate, the values obtained, nevertheless, approximate an equilibrium. This can be shown by a comparison of the K values of fructoseB-phosphate and glucose-6- phosphate. In the presence of the enzyme phosphohexoisomerase, these two esters reach equilibrium represented by the Robison-Emden ester, which consists of 2 parts of glucose phosphate and 1 part of fructose phosphate. Assuming that equal concentrations of glucose and fructose at the same pH and temperature have the same activity, then the K value of phosphorylation of fructose-6-phosphate should be twice that of glucose- 6-phosphate. This is actually the case: K for the former is 4.2 and for the latter 2.0 at 38”, pH 8.5 (Tables II and V).

When AF” is calculated according to formulas (2) and (3), the K of 1.79 for fructose-l-phosphate and 1.4 for mannose-6-phosphate corre- sponds, respectively, to AF” = - 2790 and - 2650 calories at 38”. Similarly, the K values of 2.2 and 2.3 for glucose-6-phosphate and galactose-6-phos- phate correspond to AF” = -3000 calories; the K of 0.63 for glycero- phosphate gives AF” = -2200 calories. At pH 5.8 the AF” values would be about -400 calories greater.

The discussion of the effect upon the speed of phosphorylation caused by the addition of organic phosphate of higher energy must await the re- sult of future experiments, in order to determine whether a transphos- phorylation is involved, similar to the mechanism described by Axelrod (28) *

SUMMARY

By the synthetic action of phosphatase and subsequent isolation of the esters formed, the equilibrium constant of phosphorylation was determined

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0. MEYERHOF AND H. GREEN 667

for the following ordinary ester phosphate bonds: fructose-l-phosphate, fructose-B-phosphate, fructose-l ,6-diphosphate, glucose-6-phosphate, man- nose-g-phosphate, galactose-6-phosphate, and ar-glycerophosphate. The K values for the main components of the hexose phosphates were about 2 at pH 8.5 and twice this value at pH 5.8. For a-glycerophosphate the K values were 0.63 and 1.5, respectively.

The speed of enzymatic synthesis of glycerophosphate was increased 2- to 4-fold in the presence of phosphate compounds of higher energy content. Particularly effective was phosphocreatine.

BIBLIOGRAPHY

1. Meyerhof, O., Ohlmeyer, P., and Mohle, W., Biochem. Z., 297, 113 (1938). 2. Meyerhof, O., Biol. Symposia, 6, 14 (1941). 3. Lipmann, F., Advances in Enzymol., 1, 99 (1941). 4. Meyerhof, O., Ann. New York Acad. SC., 45, 377 (1944). 5. Martland, M., and Robison, R., Biochem. J., 21, 665 (1922). 6. Roche, J., Biochem. J., 25, 1724 (1931). 7. Kay, H. D., Biochem. J., 22, 855 (1928). 8. Ohlmeyer, P., Z. physiol. Chem., 282, 1 (1944). 9. McVicar, G. A., Thesis, University of Toronto (1934).

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Otto Meyerhof and Harry GreenBIOLOGICAL ESTERS

PHOSPHATASE: I. EQUILIBRIA OF SYNTHETIC ACTION OF

1949, 178:655-667.J. Biol. Chem. 

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