Putman, S. J., Coulson, A. F. W., Farley, I. R . T., Riddleston, B., and Knowles, J. R. (1972), Biochem. J . 129, 301310. Rieder, S.V., and Rose, I. A. (1956), Fed. Proc., Fed. Am. SOC. E x p . B i d . 15. 337.
Rieder, S. V., and Rose, I. A . (19S9), J . Biol. C’hem. 234, 1007- 1010. Rose, I. A. (1962), Brookhaven Symp. Biol. 15, 293 -309. Rose, I. A., and Rieder. S. V. (1958), J . Biol. Chem. 231. 3 15-329.
Energetics of Triosephosphate Isomerase: The Appearance of Solvent Tritium in Substrate Glyceraldehyde 3-Phosphate and in Product? Susan J . Fletcher, Julia M. Herlihy, W. John Albery,; and Jeremy R . Knowles*
ZBSTRACT: When the isomerization of D-glyceraldehyde 3phosphate to dihydroxyacetone phosphate is catalyzed by triosephosphate isomerase in tritiated water, both the substrate and the product become labeled. The specific radioactivity of the product is only about 13% that of the solvent, which shows that the protonation of the enediol intermediate at C-1 (to form the enzyme-bound product dihydroxyacetone phosphate) is a kinetically significant step, and that the rate of loss of dihydroxyacetone phosphate from the enzyme is relatively fast. The
specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges protons with the medium returns to D-glyceraldehyde 3-phosphate about one-third as often as it is converted to dihydroxyacetone phosphate. These results confirm the qualitative description of the relative heights of the energy barriers in this reaction and further contribute to the quantitative analysis of the free-energy profile.
I n the previous paper (Maister et al., 1976) we have pointed out that, in the reaction catalyzed by triosephosphate isomerase, the hydrogen of the newly formed carbon-hydrogen bond is derived from the solvent and that, although neither substrate nor product can alone exchange hydrogen with the solvent, an enzyme-bound reaction intermediate can. This allowed two kinds of experiment to be performed. First, the collapse to product of the intermediate that is in isotopic equilibrium with solvent yields radioactively labeled product, the specific activity of which provides information about the product-forming step. Secondly, the partitioning of the intermediate between product and substrate can be studied by measuring the specific radioactivity of the substrate remaining after partial reaction. This experiment provides information about the energy barriers either side of the intermediate, that is, about the way in which the intermediate partitions between the twao paths open to it (back to substrate, or on to product). With dihydroxyacetone phosphate as substrate, we have seen (Maister et ai., 1976) that the discrimination against tritium i n the formation of the product (D-glyceraldehyde 3-phosphate) is 1.3-fold, which is very much smaller than that expected for a primary kinetic tritium isotope effect. This fact requires that a step in the reaction after the formation of the enzyme-glyceraldehyde phosphate complex be rate limiting. The likely situation is that the rate of loss of glyceraldehyde 3-phosphate from the enzyme-product complex is slower than thc collapse of the intermediate to give that complex. The
second type of experiment yielded the dependence of the tritium content of the remaining substrate dihydroxyacetone phosphate on the extent of the reaction. This dependence (see Figure 1 of Maister et ai., 1976) provided both qualitative and quantitative information about the partitioning of the reaction intermediate. Unlike many enzyme-catalyzed reactions, the overall equilibrium constant of the transformation catalyzed by triosephosphate isomerase allows the reaction to be studied in either direction. In the present paper we report the results of the two experiments described above. namely isotope discrimination in product formation, arid exchange vs. conversion, for the isomerase-catalyzed reaction with D-glyceraldehyde 3-phosphate as substrate. It will be apparent that new information about the catalysis is forthcoming, from the fortunate possibility of being able to study the “back” reaction as well as the “forward” reaction. Also reported here are the rquilibrium isotope fractionation factors for the two substrates i n equilibrium with tritiated water.
- ~ _ _ Hdrbdrd bniversity, Cambridge, \IdwLhu$ettF 02138 Recerr ed March 4 1976 Much of this work was done in the Dqson Perrim Ldbordtor), Lnibersitp of Oxford, England, dnd u d b ldrgel) supported by the Science Research Council under the aegis of the Oxford Enzyme Group The Phksical Chemistrl Labordtorq, Oxford OX1 342, England
’ From the Deputmenr of Cheniisrrk
5612
HIOC ~ ~ L M I I T R L Y O. L
~~~
15. NO
25, 1976
Experimental Section
Materials. Enzymes, cofactors, substrates, and other materials were as described by Herlihj et al. (1976). D L - G I ~ c eraldehyde 3-phosphate was obtained from the Sigma Chemical Co. (London, England) either as the barium salt of the diethyl acetal or as an aqueous solution of the liberated material. Methods. Measurements of radioactivitj, pH, conductivity, and ultraviolet absorbance were done as described by Herlihy et al. ( 1 976). Isomerase-Catalyzed Reactions. The transformation of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate catalyzed by triosephosphate isomerase was coupled to the reduction of dihydroxyacetone phosphate by a-glycerophos-
ENERGETICS OF TRIOSEPHOSPHATE ISOMERASE
phate dehydrogenase with NADH.I The reactions were performed in a IO-" light-path optical cuvette, in a total volume of 2.365 ml, containing: triethanolamine-HCI buffer, p H 7.6 (100 m M ) , EDTA (20.8 m M ) , N A D H (0.96 m M ) , D-glyceraldehyde 3-phosphate (0.45 m M ) , glycerophosphate dehydrogenase (0.38 mg/ml), and tritiated water (96 mCi/ml), and the reaction was initiated with triosephosphate isomerase ( 0 .I92 pglml). All the solutions. (except isomerase) were preequilibrated a t 30 O C in the cuvette. Before addition of isomerase, two samples ( I O p l ) were withdrawn for determination of the specific radioactivity of the solution. After the addition of isomerase the course of the reaction was followed by monitoring the disappearance of N A D H a t 366 nm. This wavelength (rather than the A,, at 340 nm) was used so that the initial absorbance was less than 2. An extinction coefficient for N A D H at 366 nm of 3300 M-l cm was used (Hohorst, 1956). The reaction was stopped after the desired extent of reaction had been reached, by lowering the p H to about 3.5 by the addition of 1 M HC1 (210 pl) and rapid cooling to 0 O C . Isomerase is completely inactive under these conditions (Plaut and Knowles, 1972). Separation Methods. Before separating the components of the reaction mixture, the solvent was removed by freeze-drying in order to reduce the background level of radioactivity in the column eluate and to minimize the amount of column washing required to reduce the eluting radioactivity to acceptable levels. The quenched reaction mixture was freeze-dried in vacuo as described by Herlihy et al. (1976). The residue was dissolved in 0.16 m M HCI and applied to a column ( I O cm X 0.76 cm2) of Dowex 1 (8% cross-linked, 200-400 mesh) that had been converted to the bisulfite form by treatment with saturated sodium bisulfite (100 ml), followed by water (250 ml) and 0.16 m M HCI (250 ml). After application of the reaction mixture, the'column was washed with 0.16 m M HCI until the radioactivity of the eluate reached background levels. A nonlinear gradient elution of 0.16 m M HCI (40 ml) to 1.0 M HCI (20 ml) resulted in the separation of glyceraldehyde 3-phosphate and sn-glycerol 3-phosphate. Fractions of ca. 1 ml were col1ected. Determination of the Tritium Fractionation Factors for Dihydroxyacetone Phosphate (@,) a n d Glyceraldehyde 3Phosphate ( @ p ) . Dihydroxyacetone phosphate (20 m M ) was incubated in triethanolamine-HCI buffer, p H 7.4 (100 m M ) , a t 30 O C for 2 h in the presence of triosephosphate isomerase (12.5 pg/ml) and tritiated water ( I O pl/ml of 5 Ci/ml). Duplicate samples (25 pl) were taken before and after the incubation and diluted into deionized water (500 ml) from which portions (50 PI) were taken for scintillation counting. After the incubation, 1 M HCI (1 50 pl) was added and the mixture was frozen and freeze-dried in a closed system (see Herlihy et al., 1976) to remove most of the tritiated water. The residue was dissolved in water ( 5 ml) and passed through a column (3.0 cm X 0.4 cm2) of Dowex 50 ( H + form) to remove isomerase. In experiments where the fractionation factors for the separated triose phosphates were measured (rather than the composite fractionation factor for the 96% of dihydroxyacetone phosphate 4% glyceraldehyde 3-phosphate), the glyceraldehyde 3-phosphate was converted enzymically to 3-phosphoglycerate to facilitate separation from dihydroxyacetone phosphate. The pH of the eluate containing the triose phosphates was raised to ca. 7 with aqueous ammonia, and the following reagents were added: triethanolamine-HCI buffer,
+
'
Abbreviations used: NAD+, nicotinamide adenine dinucleotide; NADH, reduced NAD+; EDTA, ethylenediaminetetraacetic acid.
300
r-----l 1 A
0.3
F r a c t i o n Number
1 : Separation of glycerol phosphate and glyceraldehyde 3phosphate on Dowex 1 (HS03-). The column was eluted with an HCI gradient (pH 3.8 to 0). ( 0 )Glycerol phosphate; (H) glyceraldehyde 3phosphate; (0)radioactivity. For experimental details, see the text. FIGURE
p H 7.4 (200 m M ; 2 ml), containing EDTA (10 m M ) , sodium arsenate ( I O m M ) , N A D + (7 mg), and isomerase-free glyceraldehyde-phosphate dehydrogenase (500 pl of a solution containing 0.5 mg/ml). When the absorbance at 340 nm had reached a constant value, the p H of the mixture was adjusted to 4 with 1 M HCI and the solution passed through a column of Dowex 50 (as above) to remove the enzyme. The eluate and washings from this column (40 ml) were diluted to give a conductivity of less than 500 pS, and the pH of the solution was adjusted to 7 with aqueous ammonia (ca. 1 M). The resulting solution was subjected to chromatography on a column (20 cm X 1.5 cm2) of DEAE-cellulose (DE 52) equilibrated with triethanolamine-HCI buffer, p H 7.35 (5 m M ) , a t 4 "C. The column was washed with this buffer and then eluted with a linear concentration gradient (80 ml 80 ml) of 5 m M to 300 m M triethanolamine-HCI buffer, pH 7.35. Fractions (1.2 ml) were collected and acidified immediately with 1 M HCI. Samples (50 pl) were taken for scintillation counting, and portions (200 pl) for assay of dihydroxyacetone phosphate and of 3-phosphoglycerate (as described in Herlihy et al. (1 976)). In some experiments, the mixture of triose phosphates was not separated, but taken directly for scintillation counting and assay of dihydroxyacetone phosphate (when an approximate value for is obtained). Determination of Specific Radioactivity. D-Glyceraldehyde 3-phosphate was assayed by conversion to dihydroxyacetone phosphate and reduction to glycerol phosphate, using isomerase and glycerophosphate dehydrogenase. The assay solution contained 200 m M triethanolamine-HC1 buffer, pH 7.6, N A D H (0.28 m M ) , glyceraldehyde 3-phosphate (200 pl sample), in a final volume of 2.5 15 ml. Glycerophosphate dehydrogenase ( I O pl of a solution of 2 mg/ml) was added and the reaction initiated with triosephosphate isomerase (5 pi of a solution of 1 mg/ml). The absorbance change at 340 nm was monitored, an extinction coefficient of 6220 M-' cm a t this wavelength being assumed (Horecker and Kornberg, 1948). To check if any bisulfite in the fractions of glyceraldehyde phosphate could upset the assay (by reaction with NAD+), N A D + was added to an assay mixture before initiation of the reaction by the addition of the enzymes. N o increase in absorbance a t 340 nm was observed. sn-Glycerol 3-phosphate was assayed by oxidation to
+
BIOCHEMISTRY.VOL. 15,
ivo. 2 5 , 1 9 7 6
5613
FLETCHER
ET A1
rABi.1: I : The Dependence of the Incorporation of Tritium into the C-2 Position of Remaining Glyceraldehyde 3-Phosphate on the Elxtcnt of t h e Isomerase-Catalyzed Reaction.
Fractional Extent of Reaction ('
Spec. Radioact. of Solvent (cpm/uatom)
Spec. Radioact. of Remaining Glyceraldehyde 3-Phosphateh (cpm/w")
Glyceraldehyde 3-Phosphat~'
(1 - r )
(x)
(P)
(P/X)
0.30 f 0.02 0.50 f 0.02 0.60 f 0.02 0.62 f 0.02 0.70 f 0.02 0.70 f 0.02 0.81 f 0.03 0.90 f 0.02
475 600 420 800 508 600 369 000 407 700 233 600 298 000 368 000
73 640 94 250
0.I55 f 0.0 I 0.22 f 0.02
I57 150 129 000 142 680 105 000 I33 000 160 070
0.31 f 0.03
Isotopic Content of Remaining B,,'
I'
0.34 f 0.05
1.18 1.13 1.14 1.19 l.ll I.lh 1.12
0.435 f 0.05
1.08
0.35 i= 0.03
0.35 f 0.04 0.45 0.05
' I Errors quoted are estimates. These values represent the mean values for the specific radioactivitiesof the fractions containing gljceraldehyde 3-phosphate. As a fraction of that of the solvent, i.e. (specific radioactivity of remaining glyceraldehyde 3-phosphate)/(specific radioactbit) of solvent). The errors quoted are estimates. From eq 3, for u = 2.5. Mean value of Bh'. 1 . I 4 f 0.01,
The Incorporatian of Tritium into Product sn-Glycerol 3-Phosphate during the Isomerase-Cat~ii!,zedReaction o l Glyceraldehyde 3-Phosphate. T A Z H L -11: E
Spec. Radioact. of Solvent (cpm/patom)
Spec. Radioact. of Glycerol Phosphateh (cpm/w")
Isotopic
for
Content of Product'
Incomplete Exchange''
(1 - r )
(X)
(SI
(S/X)
0.62 f 0.02 0.70 f 0.02 0.81 f 0.02
369 000 233 600 298 000
42 440
0.12 f 0.01 0.1 I i= 0.01 0.15 f 0.01
Fractional Extent of Reactionu
25 700
44 700
Lorr.
((.I
Bx' '
0.005 0.004
0. I 2 5 0.1 I4
0.003
0. I 5 3
' I Errors quoted are estimates. I/ These values represent the mean values for the specific radioactivities of t h e fractions containing glycerol phosphate. This represents the tritium content of product glycerol phosphate compared with t h a t of the solvent and is (specific radioactivit) of product)/(specific radioactivity of solvent). The errors quoted are estimates. See text.
