Mar 15, 2018 - hypothetical ideal solution of unit molality. An approx- imate result (-14 f 5 kJ*molâ) was obtained for the enthalpy of isomerization of ribulose 5- ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 263,No.8. Issue of March 15,pp. 3664-3669, 1988 Printed in U.S.A.
Thermodynamics of Isomerization ReactionsInvolving Sugar Phosphates* (Received for publication, October 19, 1987)
Yadu B. Tewari, David K. Steckler, and Robert N. Goldberg From the Chemical Thermodynamics Division, National Bureau of Standards, Gaithersburg,Maryland 20899
The thermodynamics of isomerization reactions involving sugar phosphateshave been studied using heatconduction microcalorimetry. For the process glucose 6-phosphate’- (aqueous) = fructose 6-phosphate’(aqueous), K = 0.285 f 0.004, AGO = 3.11 f 0.04 kJ*mol”, A P = 11.7 f 0.2 kJ-mol”, and A C = 44 f 11 J*mol”-K” at 298.15 K. For the process mannose 6-phosphate’- (aqueous) = fructose 6-phosphate2(aqueous), K = 0.99 f 0.05, AGO = 0.025 f 0.13 kJ*mol“, = 8.46 f 0.2 kJ*mol“, and A C = 38 f 25 J*mol”*K“ at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. An approximate result (-14 f 5 kJ*mol”) was obtained for the enthalpy of isomerization of ribulose 5-phosphate (aqueous) to ribose 5-phosphate (aqueous). The data from the literature on isomerization reactions involving sugar phosphates have been summarized, adjusted to a common reference state, and examined for trends and relationships to each other and to other thermodynamic measurements. Estimates are made for thermochemical parameters to predict the state of equilibrium of the several isomerizations considered herein.
The isomerizations of glucose 6-phosphate to fructose 6phosphate, of mannose 6-phosphate to fructose 6-phosphate, and of ribose 5-phosphate to ribulose 5-phosphate areimportant reactions occurring in metabolic processes. These three reactions are catalyzed, respectively, by phosphoglucoisomerase (EC 5.3.1.9), phosphomannose isomerase (EC 5.3.1.8), and phosphoriboisomerase (EC 5.3.1.6). The first reaction is a key step in the reactions of glycolysis by which living organisms utilize the energy available in carbohydrates. These reactions are also representative of a broad class of known or possible reactions involving the interconversions of sugar phosphates. Although equilibrium data have been reported for all three of these reactions (1-12), there does not appear to be any calorimetric data available for any of these processes. The principal aim of this study was to perform direct heat measurements to determine the enthalpy changes for the above three isomerizations. Since these reactions have extents of reaction ranging from 20 to 80% amount of substance reacted, it was also possible to calculate equilibrium constants from the calorimetric results (13). These measurements are interpreted using a thermodynamic modelwhich includes effects from proton and metal-ion binding to both reactants and products. The model also has predictive value and allows one to calculate equilibrium constants and enthalpies of reaction under conditions at which measurements have not
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been made. The results obtained herein are also related to other phosphorylation reactions via thermochemical networks, which are useful for discovering possible systematic errors in the measurements and in establishing the accuracy of the thermodynamic parameters for each of the processes in the network. EXPERIMENTALPROCEDURES
Tris, HCl, and MgCl, were obtained from Fisher. All other chemicals were from Sigma. Phosphoglucose isomerase and phosphoribose isomerase were received in lyophilized form. Phosphomannose isomerase was contained in a 3.2 M (NH4),S04 0.5 M K,PO, solution. This enzyme solution was dialyzed against Tris/HCl buffer containing MgC1,. Karl Fischer analysis was used to determine the amounts of water in each of the substrates. The results, given in mass percent of water for each of the substrates, are: Glu-6-P, 17.8 f 1.8%;Fru-6P, 10.6 0.7%; Man-6-P, 12.6 f 0.9%; Rib-5-P, 13.7 f 2.0%; and Ru-5-P,’ 3.01 f 0.07%. The purities of the sugar phosphates were determined by alkaline phosphatase hydrolysis to the corresponding sugar and inorganic phosphate. The sugars were then analyzed using the HPLC methods previously described (14). This procedure was used due to thedifficulties in obtaining direct separations of the sugar phosphates themselves. The results, given in mass percent of the impurity are: Glu-6-P, 0.8% Fru-6-P; and Fru-6-P, 0.26% Glu-6-P. Mannose 6-phosphate and ribose 5-phosphate showed no detectable impurities. According to the vendor, the ribulose 5-phosphate contained 2.4% ribose 5-phosphate, 0.7% xylulose 5-phosphate, 0.4% methanol, and 0.05% triol phosphate. The amounts of these impurities were confirmed in our laboratory. Similar HPLC analyses were performed on the reaction mixtures to determine if there were any side reactions occurring during the isomerization processes. For the conversions of glucose 6-phosphate to fructose 6-phosphate and of fructose 5-phosphate to mannose 6phosphate, there was no evidence of any side reactions. However, for the isomerization of ribose 5-phosphate to ribulose 5-phosphate, a peak attributable to xylulosewasobserved.Also, the calorimetric thermogram was not a single peak, but was one characteristic of two separate reactions occurring. Therefore, we conclude that the ribose5-phosphate isomerase contained some ~-ribulose-5-phosphate3epimerase (EC 5.1.3.1),which converted ribulose 5-phosphate to xylulose 5-phosphate. Because of this problem, only an approximate result for this isomerization could be obtained. The calorimetric procedures have been described previously (15, 16). Measurements of reaction heat were performed by mixing, in the calorimeter, a substrate solution and an enzyme solution. The substrate solution was prepared by dissolving a known amount of the substrate (e.g. glucose 6-phosphate, fructose 6-phosphate, etc.) in a buffer solution containing a known concentration ofMgCl,. The enzyme solution was prepared by adding the same buffer solution to the lyophilized enzymes. “Blank”heat effects were measured by mixing the enzyme solution with a substratesolution which contained only the buffer with MgCI,. These blank heat effects ranged from 0.05 to 0.33 mJ. They were applied as small corrections (
