THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 236, No. 12, December 1961

Printed in U.&A

Intramolecular Hydrogen Transfer in the

Phosphoglucose Isomerase Reaction

IRWIN A. ROSE AND EDWARD L. O’CONNELL

From the Department of Biochemistry, Yale University, New Haven, Connecticut

(Received for publication, May 4, 1961)

The conversion of fructose 6-phosphate to glucose 6-phosphate catalyzed by phosphoglucose isomerase in DzO is known to result in the incorporation of deuterium at C-2 (1). From the fact that the deuterium content of the product was close to that of the medium it has been assumed that direct transfer of hydrogen from C-l of fructose 6-phosphate did not occur. The experi- ments reported will show that such a transfer does occur, that under certain conditions the extent of direct transfer may greatly exceed the incorporation from the medium, and that the transfer represents a net shift of H from C-l to C-2 of the same substrate molecule. The data are interpreted iu terms of the usually proposed “enediol” mechanism for keto-aldehyde isom- erism and are thought to provide information concerning the basic group on the enzyme which attacks the a-hydrogen of the substrate.

EXPERIMENTAL PROCEDURE

Phosphoglucose isomerase preparations from three sources were examined in these studies. The preparation of Tsuboi et al. (2) from human red blood cells was taken through step D, specific activity 9 units per mg of protein. The preparation of Slein et al. (3) from rabbit muscle was taken through step 7, specific activity 80 units per mg of protein. Crystalline phos- phoglucose isomerase from yeast was obtained from Boehringer and Son, specific activity 390 units per mg of protein. The stated specific activities are those obtained at the time of prepara- tion or purchase. A unit of enzyme is defined as that amount which results in the production of TPNH at a rate of 1 pmole per miu in the following assay: A total volume of 1 ml contains 0.5 pmole of fructose-6-P, 5 pmoles of MgC12, 100 pmoles of triethanolamine-Cl buffer, pH 8.0, 0.5 pmole of TPN, and at least 0.5 unit of glucose-6-P dehydrogenase. The rate of reaction was proportional to the amount of isomerase in the range 0.002 to 0.02 unit. A unit of the dehydrogenase is defined in the same way but with 0.2 pmole of glucose-6-P in the assay instead of fructose-6-P. No reduction of TPN was found in such an assay with mannose-6-P instead of fructose-6-P, indicating the absence of mannose-P isomerase in these preparations. Furthermore, added glucose-6-P dehydrogenase (Boehringer) was necessary to obtain TPN reduction, indicating the absence of this enzyme in the three preparations of phosphoglucose isomerase.

Glucose-6-P was analyzed spectrophotometrically with glu- cose-6-P dehydrogenase. Fructose-6-P was determined by the difference between the amount of TPN reduced in the presence of phosphoglucose isomerase plus glucose-6-P dehydrogenase and that produced with glucose-6-P dehydrogenase alone. In addi-

tion, fructose-6-P was determined by the less specific calorimetric assay for keto-sugars (4).

Deuterated (and tritiated) forms of glucose-6-P and fructose- 6-P were prepared as described in the text. When these com- pounds also contained C14, glucose-l-U4 was first converted to glucose-6-P with purified yeast hexokinase and ATP, and this product was incubated in DzO with isomerase as described for the usual preparation of the deuterated compounds.

Periodate oxidation of 6-P-gluconate was done in 0.05 N

H2S04 in order to liiit the further oxidation of the glyoxylic acid formed. The fructose-6-P was oxidized with NaI04 at neutral pH. Glycolate and glyoxylate were isolated by ion exchange chromatography and assayed calorimetrically (5, 6). Radio- activity was determined by liquid scintillation counting (7).

RESULTS

Preparation of Tritiated and Deuterated Substrates-In order to attain isotopic equilibration between the reactants and the tritiated water of an incubation medium it was necessary to use prolonged incubations. A mixture containing 100 mc of tritiated water and 200 pmoles of glucose-6-P, free of barium, was adjusted to pH 8 in a total volume of 0.50 ml. Muscle isomerase (16 units) was added and the solution placed at 25’ for 10 hours. The solution was evaporated in a vacuum and the residue was dissolved in 1 ml of 0.2 N HCl and dried again. The residue was dissolved, neutralized iu the cold with dilute triethanolamine, and treated with a slight excess of barium acetate to precipitate the glucose-6-P as the barium salt of the heptahydrate. After allowing several hours for crystallization, the precipitate was washed with cold water, dissolved in dilute HCl, carefully neutralized in the cold and allowed to recrystallize. The super- natant solution from the first crystallization was used for the preparation of tritiated fructose-6-P which was fractionated first on Dowex l-borate (8) and then after removal of borate (8), refractionated on Dowex 1 (Cl-) by elution with 0.025 N HCl. The specific activities of the isolated compounds and water were: Glucose-B-P, 83,000 c.p.m. per pmole; fructose-6-P, 83,500 c.p.m. per pmole; and water, 86,500 c.p.m. per yatom of hydrogen. That only one hydrogen becomes affixed to the substrates is in agreement with earlier results (1) with phosphoglucose isomerase and is consistent with the complete specificity of attack on only one of the C-l hydrogens of fructose-6-P (9).

When preparations of the deuterated compounds were made, a trace of tritiated water was included with the DzO so that the attainment of deuterium equilibration could be established by means of the tritium tracer. Control experiments were per-

3086

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December 1961 I. A. Rose and E. L. O’Connell 3087

formed to establish that there was no decrease in the specific TABLE I activity of the tritiated compounds when treated by the tech- Ratio of exchange to transfer niques used in their purification. Fructose-B-P,l-T (about 0.15 mM, 83,000 c.p.m./pmole), TPN

The following evidence was obtained to indicate that the tritium was located only in the C-2 position of the glucose-6-P. The glucose-6-P was oxidized to 6-phosphogluconate (10) and this was converted to glyoxylate by periodate oxidation. All of the three compounds had the same specific activity to within 5%. A direct periodate oxidation of the tritiated fructose-6-P gave rise to glycolic acid which had the same specific activity as the starting compound, thus establishing that all of the tritium was in the C-l position of the fructose-6-P.

Demonstration of T&urn Transfer in Going from Fructose-B-P JO Glucose-B-P-A mixture was made containing glucose-6-P (26 &moles) and fructose-6-P,l-T (138 pmoles, 207,000 c.p.m.) as barium salts and adjusted to pH 8.0 with triethanolamine, in a total volume of 2.5 ml. To 0.5 ml of this was added 50 I.cmoles -of the barium salt of glucose-6-P dissolved in dilute acid. The barium salt of glucose-6-P was crystallized from the neutralized solution and recrystallized. The radioactivity in this material was used as a measure of the content of tritium in the glucose-6-P of the original mixture. The total number of such counts in the remaining 2.0 ml of solution would have been 590 cpm of the total 166,000 c.p.m. present. After this solution had begun to yield crystals of the barium salt of glucose-6-P in the cold, 1.6 units of muscle isomerase were added and incubation was con- tinued for 2 hours at 25” after which the crystals formed were recovered, dissolved, and reformed. The specific activity ob- tained was 645 c.p.m. per pmole and 45 pmoles were recovered. The supernatant fluid and wash of the crystals were pooled and put through a Dowex I-(Cl-) column. Of the 166,000 c.p.m. present, 84,000 c.p.m. were not retained by the resin. If the 82,000 c.p.m. not exchanged are assumed to be distributed among the 131 pmoles of glucose-6-P that could have been formed in the incubation, the predicted specific activity would be 626 c.p.m. per pmole. The glucose-6-P was next oxidized with IZ to 6-phosphogluconate (10). This was isolated as the barium salt insoluble in 66% ethanol and had a specific activity of 645 c.p.m. per pmole. This was in turn oxidized with HI04 and the glyoxylic acid recovered was found to have a specific activity of 635 c.p.m. per pmole. From these data it may be concluded that the fructose-6-P was converted to glucose-6-P with the loss of about half the tritium to the medium and the transfer of the remainder to C-2 of the product.

(0.6 mM), MgClz (4 mM), triethanolamine-Cl- (20 mM, pH 7.7) and glucose-6-P dehydrogenase (1.7 units per ml) were incubated at 27” with about 0.01 unit phosphoglucose isomerase from various sources in three experiments : (1) human red cells, (2) rabbit mus- cle, and (3) yeast. Two samples of 0.5 ml were removed at 0 minutes and after various extents of reaction as determined by TPNH formation. To one of each pair was added 3 pmoles of HgClt to stop the reaction and this sample was used to determine counts not retained by Dowex 1 (Cl-) on a short column, i.e., exchanged counts. The other sample was acidified to inactivate the glucose-6-P dehydrogenase, neutralized, and treated with 1.6 units of muscle isomerase at 37” for 10 minutes. It was then put on a Dowex 1 (Cl-) column to determine, by acid elution, counts retained b.y the resin, i.e. transferred counts.

TPNH formed

Extent of et reactiol

Ikitium in water ”

1

Ikitium in product

-

._ ! n

-

- I

.-

Sxchange transfer

zpmoles/m, % 32.4 23 64.8 46

141.8 100

lZp?ZOl~S/~l

5.5 12 65.6

npwles/ml

5.8 12.3 66.6

0.95 0.97 0.98

32 24.6 5.4 64 49.2 12

130 100 54.7

7 0.77 15 0.80 72.4 0.76

32.4 19 6.5 64.8 38 13.5

168 100 111

4.3 7.3

56.5

1.50 1.80 1.95

reaction was found, on ion exchange chromatography (Dowex l- (Cl-), eluted qiith 0.025 N HCl) to give a single symmetrical radioactive peak, separated from fructose-6-P, TPN, and the acid degradation product of TPNH. This peak contained all of the radioactivity retained on the column and assayed as 6-P- gluconate with the purified dehydrogenase (11). In Table I are presented data for an incubation with the red cell enzyme. The amount of tritium exchanged is almost the same as that transferred. Similar experiments using enzymes from muscle and yeast (Experiments 2 and 3) also showed appreciable amounts of tritium transfer. However, the ratio of exchange to transfer differed significantly in the three experiments.

Determination of Relative Magnitude of Exchange and Transfer -To study the comparative rates of the two processes the follow- ing procedure was used. Fructose-6-P containing T at C-l was incubated with rate-limiting amounts of isomerase and excess glucose-6-P dehydrogenase plus TPN. In this way, formed glucose-6-P was immediately converted to 6-P-gluconic acid in which form it was no longer subject to detritiation. Two sam- ples were removed at each of several percentages of complete reaction. One such sample was used to determine tritium ex- changed into water as the counts not retained by Dowex 1 (Cl-). The other was treated with acid to inactivate the enzymes, neutralized, and excess isomerase was added to detritiate any fructose-6-P remaining. The radioactivity retained by a Dowex 1 (Cl-) column after such a treatment was a measure of the counts in 6-P-gluconate and hence of the tritium transferred. This was shown to be the case since a sample removed at 100%

It will be noted from Table I that the reactivity of the tritiated substrate either toward exchange with the medium or transfer is much less than that of the normal fructose-6-P in the over-all reaction. Thus in Experiment 1, at a time when 23% of the fructose-6-P had formed product, only 8% of the fructose-6-P, 1-T had reacted in both exchange and transfer. This apparent discrimination decreases as the remaining substrate becomes enriched in the tritiated species. Thus, toward the end of the reaction, the rate of appearance of tritium in water and product increases. This is shown in the experiment of Fig. 1 in which the product of the isomerase reaction with fructose-6-P, 1-T is trapped by the glucose-6-P dehydrogenase reaction, as in Table I. In this figure, the fraction of those counts appearing in the water is plotted against the fraction of net reaction. Eventually only 46% of the total counts in the experiment are lost by ex- change, but this value is taken as 100% for this figure. The data fit best a line plotted from the calculations of Lietzke and

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3088 Phosphoglucose Isomer-use Reaction Vol. 236, No. 12

I o-

9-

w .a- ci z 9 .7-

E -J 6-

4-

ture range 0 to 60 (Table II). As the temperature is lowered, a smaller portion of the tritium is lost by exchange, being only 17 Y. at 0”. Above 60” enzyme inactivation is too rapid to allow accurate measurements. The increased proportion of exchange at higher temperatures is not the result of thermally activated enolization of the substrate as shown by nonenzymatic control incubations. In fact the tritiated substrates do not undergo detritiation to any significant estent in this temperature range and at pH values up to 10. A linear plot of l/T vs. log (ex- change/transfer) is obtained from these data (Fig. 2), from which it would seem that an approach to complete exchange would require much higher temperatures. At 60” about 75% of the tritium is lost by exchange. From the slope of this plot one ob- tains a value which is the difference between the activation energies of the two processes, exchange and transfer, 8000 cal per mole. Also shown in Fig. 2 is an Arhennius plot for the net reaction under similar conditions. It is perhaps fortuitous that the activation energy for the reaction is also 8000 cal per mole.

Deuterium Isotope Efects in Net Reaction-The observation of discrimination against the tritiated substrate (Fig. 1) led to an examination of the deuterated substrates for an isotope effect.

0 .I .2 3 .4 5 6 7 6 .9 1.0 With the use of the muscle isomerase, rates were measured at concentrations of substrate much greater than the K, values

FRACTION OF NET REACTION

FIG. 1. Comparison of the fraction of exchange with extent of the net reaction fructose-6-P to glucose-6-P. The incubation contained in 10 ml: fructose-6-P,l-T (1.3 pmoles), TPN (2.5 pmoles), MgClz (50 rmoles), triethanolamine, pH 8.0 (200 pmoles), glucose-6-P dehydrogenase (2 units), and phosphoglucose isomer- ase of muscle (0.0065 unit). The incubation was placed at 25” and optical density changes at 340 mp were recorded as a measure of the net reaction. Samples were taken at several time intervals for the determination of exchanged radioactivity. To obtain the value for eventual exchange, 0.1 unit of isomerase was added to 1 ml of incubation solution in which the appeaance of TPNH seemed complete. The solid line is derived from theoretical curves (12) for an isotope effect, kT/kH = 0.36. The dots indicate the experimental points.

TABLE II Effect of temperature on ratio of exchange to transfer

Incubations, similar to those described in Table I, using the muscle enzyme were set up at various temperatures. The length of each incubation period was determined by the time required to convert at least 50% of the fructose-6-P to glucose-6-P and was not the same at each temperature.

Temperature Tritium exchanged Tritium Exchange transferred transfer

OC c.fu?L/ml C.@%/?Xl

0 1304 6480 0.205 5 968 3264 0.296

15 2800 6000 0.451 25 4480 5292 0.850 37 5568 4236 1.31 50 7736 2256 3.44 60 4320 1400 3.17

Collins (12) for an isotope effect kT/lcH = 0.36. From the shape of this curve it is readily seen that at large percentages of reac- tion, the amount of tritium exchange will be very sensitive to small variations in the extent of product formation.

Effect of Temperature 012 Ratio of Exchange to Transjer-Exper- iments similar to those in Table I were carried out in the tempera-

-

-.4-

-.5 -

--I 9

- -2.0

--2 I

--22

--23 5

0

--24 ‘;=

0 0

--25 h

f --2x c

r;l

- --2.7

- -2.8

--29

-.6 - - -3.0

-.7L 0 .

3.0 3.1 32 3.3 3.4 35 3.6 3.7

(f ) x 103

FIG. 2. Arrhenius temperature dependence of the exchange: transfer ratio and of the rate of the net reaction fructose-6-P to glucose-6-P. The values for the exchange:transfer ratios (+---a) are those given in Table II. The values for the rate of the net, reaction (O--O) are expressed as change in absorb- ancy per minute (A O.D. per minute) with the conditions of the standard assay for the isomerase in which initial rates are deter- mined spectrophotometrically. The muscle enzyme was used.

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December 1961 I. A. Rose and E. L. O’Connell 3089

(10) so that the results obtained represent isotope effects on the masimal velocities. The reaction, fructose-6-P to glucose-6-P, was followed in the presence of excess glucose-6-P dehydrogenase measuring TPN reduction at 340 rnl.c. Deuterated fructose-6-P reacted at 45% the rate of the normal substrate. The reaction, glucose-6-P to fructose-6-P was followed with the calorimetric test (4). The deuterated substrate reacted at 50% the rate of the natural substrate.

It was of interest to determine the rate of reaction of glucose- 6-P) 2-D in the presence of an excess of normal glucose-6-P. To do this glucose-6-P ,2-D, l-U4 was mixed with 26 times the amount of glucose-6-P. The mixture was recrystallized 4 times as the barium salt to reduce the contamination with fruc- tose-6-P to less than O.l%, as determined by enzymatic assay. This glucose-6-P was then incubated with muscle enzyme in the presence of borate (13), which was added to shift the equilibrium ratio fructose-6-P:glucose-6-P from 0.3 to 0.79 in these experi- ments. Samples were removed during the course of the reaction and fructose-6-P isolated and its specific activity determined. It was of interest to compare the specific activity of fructose-6-P obtained at early times with that of the glucose-6-P in order to determine the relative rates of reaction of the two species when both were present. As seen from Table III, the normal species reacted on the average at 1.86 times the rate of the deuterated glucose-6-P when the two forms were in competition for the enzyme.

Evidence for Intramolecular Character of Transfer Reaction- The observed difference in kinetic behavior of normal and deuterated glucose-6-P in the isomerase reaction is the basis for the method used to show that fructose-6-P, l-D, l-Cl4 (inter- nally doubly labeled) in the presence of a large amount of normal fructose-6-P, is converted to glucose-6-P in which Cl4 and deu- terium are distributed as would be predicted for intramolecular transfer. If, as shown in Table III, glucose-6-P,ZD reacts as an average at l/1.86 = 0.538 times the rate of the normal sub- strate it is only necessary to know the specific radioactivity of fructose-6-P formed during early stages of the isomerase reaction to know the distribution of Cl4 between normal and deuterated substrates. The equations for such a calculation are now de- rived.

Normal and deuterated substrates of concentrations S and S’ form a mixture of molecules in which Cl4 is distributed to give initial specific activities A, and A,,. The two substrates form product at the rate v and v’ when they are present in the mixture. The expression for the ratio v/v’ has been derived previously (14) in terms of the kinetic constants for each substrate alone, equation (1). From this it follows that the quantity vS’/v’S is a constant, here called 2. The specific activity of the product

(1)

formed during the early phase of the reaction, Ai, is given in equation (2). Multiplying numerator and denominator by

Ai = v’A: + vA,

a 0’ + v (2)

1+j

TABLE III

Competition between glucose-6-P and glucose-6-P&D in isomerase reaction

Glucose-6-P,2-D,l-Cl* was diluted 27-fold with glucose-6-P and recrystallized 4 times, specific activity 890 c.p.m. per pmole. Two equivalent experiments, using this material, were run on different occasions: Experiment 1. 129 hmoles of glucose-6-P and 920 pmoles of Na2B40v, adjusted to pH 8.0 in 12 ml were incu- bated at 37” with 1 unit of muscle isomerase. Samples were taken at 7 and 14 minutes as noted and the content of fructose-6-P was determined calorimetrically. To the remaining incubation (3 ml), 2 units of enzyme were added and after 30 minutes, sufficient to equilibrate the reactant and product isotopically, 44% of the glucose-6-P was found as fructose-6-P. Experiment 2 was similar to above. Fructose-6-P was isolated from all samples by ion ex- change methods.

Experiment

min % c.~.m./pmole 1 7 7.5 497 1.79

14 12.8 445 2.00 45 44* 890

2 10 13 491 1.81 20 26 484 1.84

* 44% represents the amount of glucose-6-P found as fructose- 6-P after equilibration.

Ai = A,, ; + ZA,

$+Z (3)

The value of 2 is determined in the experiments of Table III in which A, = 0, S/S = l/26 and the final specific activity of the product, Ar, is given by AI = S’A,‘/(S + S’). Applying these values to equation (3) and by using the average experimental value for the ratio A//A; = 1.86, the characteristic constant 2 = 1.89 is obtained.

In the general case, in which the radioactivity is present in both S and S’ it is possible to determine its distribution from the experimental ratio At/A,. Let x = the fraction of Cl4 present in S’ at the start of the reaction. Then,

8’A.J x = S’A., + SA, ’

A 8’

= x-A,.@ + S’)

S’ ’

and

A *

= (1 - x).A,.(S + S’) S (4)

Inserting these values of A, and A,, in the general equation (3), results in the final equation (4).

x=~($+z)-z(l+~) (4) ( > 1 + 5 (1 - Z)

S/S gives equation (3).

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3090 Phosphoglucose Isomerase Reaction Vol. 236, No. 12

Table IV describes the results of two experiments in which fructose-6-P) l-D, l-CY4, diluted 23-fold with normal fructose-6-P was first converted, at 20” and O’, to glucose-6-P in the presence of barium ion, in order to trap the product; and the purified glucose-6-P was then analyzed to determine the distribution of Cl4 between normal and deuterated species. The values of z obtained are less than 1 indicating the presence of some Cl4 in the nondeuterated species; however, this is an amount which would be expected from the data on exchange of tritium from fructose-6-P) 1-T during the enzymatic reaction at these tem- peratures. The distribution of Cl4 to be expected if the transfer of deuterium were intermolecular would be given by 2 = l/23 = 0.044.

DISCUSSION

The demonstration of transfer of hydrogen between reactant and product in reactions in an aqueous environment is generally interpreted as evidence for the participation of a hydride ion or a hydrogen atom as the transferred species. The occurrence of exchange with hydrogen ions of the medium is not consistent with either of these and would usually be assumed to indicate a second, independent mechanism. With enzymatic catalyses, however, the occurrence of parallel reactions catalyzed by a single enzyme seems improbable. The possibility that the hy- drogen transferred may be bound to a group on the protein at some stage of the reaction might provide the solution of this conflict consistent with either a proton shift or a hydride shift. Proton transfer to a basic group on the enzyme would provide a mechanism comparable to that proposed for the base catalyzed Lobry de Bruyn transformation (15). Exchange would then be assumed to occur between Hz0 and the conjugate acid of the C-H bond-rupturing base. This exchange may not be in- stantaneous or it may be that free access of the site to water is somewhat limited. The intermediate would contain the substrate as an enediol. Since full stereospeci6city of tritium labeling from the medium is found in fructose-6-P it is clear that any enzyme-enediol intermediate would not be free to dissociate. Thus, the enediol may constitute a steric barrier to the diffusion of water, as may a particular folding of the protein. The con- jugate acid group on the enzyme must then transfer its hydrogen, part of which will have exchanged, to either C-l or C-2 of the bound enediol.

There is, at present, no data that would allow one to distin- guish between this enediol mechanism and an oxidation-reduc- tion mechanism in which a group on the isomerase molecule is reduced by the hydride ion originating from the substrate. In such a mechanism, the hydrogen would undergo limited exchange with the water before being transferred to the oxidized inter- mediate, glucosone-6-P, and either regenerate the substrate or produce the product. One may prefer the enediol mechanism since it is a natural consequence of polarization of the ar-carbonyl group and it provides a common basis for considering other examples of a-hydrogen activation such as the aldolase condensa- tion and pyruvate-phosphokinase reactions which involve loss of a proton without oxidation.

If we tentatively accept the enediol mechanism one can draw certain conclusions regarding the geometry of the enediol. As suggested in an earlier report (9), the available data concerning the stereospecificities of the four isomerases: phosphoglucose isomerase, phosphopentose isomerase, triose-P isomerase, and phosphomannose isomerase are consistent with a common

geometry for the suggested enediol bound to the enzyme. If the hydrogens that will occupy C-l or C-2 approach from oppo- site sides of the plane of the enediol, the absolute configuration of the products would require that a trans-enediol be involved. If the hydrogens approach from the same side, a cis-enediol is required. The demonstration of an intramolecular hydrogen transfer can best be interpreted as attack by a single conjugate acid group on either C-l or C-2 and so requires that the inter- mediate be closer to a cis-enediol.

The experiments which demonstrate that deuterium was trans- ferred from C-l to C-2 of the same molecule rule out mechanisms. in which an intermediate, having lost its hydrogen to the enzyme, is free to dissociate from the protein and those in which the sub- strate is protonated at the carbonyl-carbon by an acid group on the enzyme at the same time that the a-hydrogen is removed by a basic group. In addition, these data provide evidence about the nature of the attacking base in the enediol mechanism. Since the enzyme concentration is much smaller than that of the sub- strate, the enzyme will have turned over many times in the course of the measured reaction. If the activating basic group of the reaction site of the enzyme were, for instance, an e-amino group of lysine, it would have acquired and lost the hydrogen of the substrate many times through the reaction sequence in equa- tion (5). Assuming free rotation of the E-NH~+ group around

H-N-H

r

H

Enz. + H-R --) 1

L

H-FH R- + H-N-H + product. (5)

Enz. J I

Enz.

the C-N bond and, therefore, an equal chance for the three hydrogens to be transferred in the second step, one would expect the amino-hydrogens to have the same deuterium content as the substrate C-2 position, namely 1 deuterium per 23 atoms in the experiments reported in Table IV. The conjugate acid derived from attack of the enzyme on a particular molecule of fructose- 6-P having both Cl4 and deuterium would have an average atom percentage excess for the three hydrogens of the resulting am- monium group of l/3 (1 + 2 * l/23) = 0.363. This dilution sets a limit of 0.363 on the fraction of Cl4 that can be associated with deuterium in the glucose-6-P formed. This calculation ignores both exchange at the conjugate acid stage and any isotope effect selecting against the transfer of deuterium relative to hydrogen in the step from the conjugate acid to the enediol. Both of these phenomena would reduce the fraction of intramolecular double- labeling further. Similar considerations pertain to a base with one exchangeable hydrogen. On the other hand, for a base witk no exchangeable atoms, the exchange must occur exclusively in the conjugate acid form and, therefore, before transfer. Con- sequently, it is necessary to correct for loss of deuterium by exchange in making this calculation. Since there is no reason for expecting the exchange: transfer ratio to be different with deuterium, the values obtained from comparable experiments with tritiated fructose-6-P are used. To provide a more direct evaluation of the effect that dilution of deuterium in the con- jugate acid would have on the early specific activity of the product, the observed data from Table IV and the values cal- culated for a base with 0, 1, and 2 equivalent hydrogens are given in Table V. The results can be interpreted best in favor of a base which has no exchangeable hydrogens, i.e. a carboxylate group or the nonprotonated nitrogen of an imidazole group.

It remains to explain the failure of Topper to observe hydrogen

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December 1961 I. A. Rose and E. L. O’Connell 3091

transfer in his experiment (1) in which fructose-6-P was incu- bated with muscle isomerase in DzO (95.7 atom per cent) in the presence of barium ions. The glucose-6-P was found to contain 0.91 atom deuterium per mole. The explanation may be that the reactants had equilibrated with the DzO before the barium salt of glucose-6-P had precipitated. Topper makes no mention of the amount of enzyme used in this experiment, which makes it impossible to evaluate this suggestion. As the present work indicates, it is important in cases in which both transfer and ex- change may be occurring to measure isotope distributions at times long before equilibrium has been reached. This has the disadvantage, when one is working with DzO-HZ0 mixtures, of allowing isotope discrimination to influence the results. This is always a problem when using tritiated water. A minimal pre- caution would seem to be the use of D- or T-substituted sub- strates to test for hydrogen transfer directly.

Similar experiments (16) with triosephosphate isomerase starting with tritiated dihydroxyacetone-P failed to reveal any transfer of tritium to the glyceraldehyde-3-P produced. It may be, however, that the intermediate conjugate acid group is much

TABLE IV Demonstration of intramolecular nature of transJer reaction In the first part of Experiment 1, a mixture was made which

contained 10 pmoles of fructose-6-P,1-D,1-C14 (220,000 c.p.m. and about 99% D in the isomerase-activated position at C-l), 218 rmoles fructose-6-P and 39 pmoles glucose-6-P all as barium salts, pH adjusted to 8.0 with triethanolamine. Crystals of Ba glucose- 6-P were permitted to form before adding 3 units of muscle isomer- ase. The suspension was incubated at 20” for 2 hours. The crys- talline product was recrystallized, barium removed from it, and the glucose-6-P used in the next part of the experiment. Incu- bate at 37” in 4.0 ml, 150 pmoles of glucose-6-P prepared above (specific activity, 765 c.p.m./pmole) with 680 pmoles of NazB407 at pH 8 and 1.6 units of muscle isomerase. Samples were taken at 30 and 100 minutes after 16.6 and 27.5yn of the glucose-6-P had been converted to fructose-6-P. Excess enzyme was added to the remainder and sufficient time allowed to achieve equilibration (33% conversion under these conditions). The samples were treated by ion exchange for isolation of fructose-6-P.

Experiment 2 was conducted in the same way with the following exceptions. The conversion to glucose-6-P was done at 0”. The glucose-6-P isolated was recrystallized 4 times and had a specific activity of 862 c.p.m. per pmole. The conditions used for its conversion to fructose-6-P were: glucose-6-P (10 mrvr), Na2BI0, (82 mM), pH 8.0, and 0.6 unit of muscle isomerase per ml. A sam- ple was taken at 14.4y0 conversion.

s - c.

-

I I

Initial Fraction of specific Fructose- Cl4 associ-

Ictivity of ~~~“,;i6,f 6-P specific ated with :lucose-6-P activity g~;-sc@-

p.m.//A?nole y. c.~.nz./jmole

765 16.6 522 0.69 27.5 670 33c 765

862 14.4 526 0.81 81c 840

-

7;

-

Zxchange y experi- mentb

%

39

16.7

a (l-z)100 b Approximated from Table II for 20” and 0’. c Samples obtained after equilibration, A,. d Corresponds to z in equation (4). Calculated using 2 = 1.89

and S’/S either 0.024 or 0.032 in Experiment 1 or 2, respectively.

TABLE V Effect of base with 0, 1, or 2 hydrogens on specific activity of

initially formed fructose-6-P The data of Table IV are tabulated again. To calculate the

early specific activity, Ag, for the case of the enzyme site with an attacking base with 0, 1, or 2 equivalent hydrogens, equation (4) is solved for A; with the values of x which would correspond to the particular model under consideration. Thus, with a base of 0, 1 or 2 hydrogens, the value of z would be 1,0.522, or 0.363, respec- tively. The existence of exchange requires that the value of z be modified in the case of the base with no protons, as discussed in the text. The correction here is dependent upon temperature and as derived from Table II would give x = 0.61 and 0.83 at 20 and 0 C, respectively.

Ai

Experiment Early spe-

cific activity of fructose-

6-P

Calculated early specific activity with a base of le hydrogen

n=o 1 n=l 1 n=2

c.).m./p%oles

545 635

524 709

c.).m./pmoles c.fLm./@iwles

1 765 522

2 840 526

more accessible for exchange with the medium in the triosephos- phate isomerase mechanism.

SUMMARY

In agreement with previous work it is found that the inter- conversion of glucose B-phosphate and fructose 6-phosphate by the enzyme phosphoglucose isomerase in tritiated water results in the incorporation of 1 atom of tritium into each of the two compounds. The implication that retention of hydrogen in going from substrate to product does not occur was not borne out since it was found that fructose 6-phosphate, 1-T formed a considerable amount of glucose 6-phosphate, 2-T. The fraction of the tritium that is retained was greatest at low temperatures.

The observation was made, using deuterated substrates, that the net reaction in either direction occurs with a primary isotopic effect. With the use of this information a method was designed for determining whether or not the transfer of hydrogen occurred within the same molecule or between substrate molecules. The hydrogen migration was found to be intramolecular. The results were interpreted in terms of an enediol mechanism in which a basic group associated with the active site of the enzyme attacks the hydrogen cr to the carbonyl group of the substrate, forming a conjugate acid-enediol intermediate which would form product by a second proton migration. Evidence bearing upon the geometry of the enediol and the nature of the basic group at the active site of the enzyme is discussed.

Acknowledgement-The assistance of Mr. Gordon F. Fair- clough, Jr., in the preparation of muscle isomerase and in the performance of some of the preliminary experiments is gratefully acknowledged.

REFERENCES

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Irwin A. Rose and Edward L. O'ConnellIntramolecular Hydrogen Transfer in the Phosphoglucose Isomerase Reaction

1961, 236:3086-3092.J. Biol. Chem. 

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