552%
adenosine triphosphate molecule, i t was essential to have some estimate of the magnitude of the rate for the competing, non-terminal hydrolysis reaction a t the ribose-phosphorus linkage. For this purpose, fructose-6-phosphoric acid (FPX) was selected as an available model compound to yield order of magnitude information on the hydrolysis rate of the primary sugar hydroxyl-phosphorus grouping. Previous report^^,^ on the hydrolysis of fructose mono- and diphosphates have indicated a greater lability toward aqueous hydrochloric acid of the 1-phosphate linkage as compared to the G-grouping,? and a rate constant3 for the relatively rapid hydrolysis of fructose-1-phosphate (in 0.1 N HCl a t 100') amounting to 1 X sec.-'. In the present work, the 6-phosphate ester hydrolysis was carried out under somewhat milder conditions, closely approximating those used in previous kinetic work" on the acid-catalyzed hydrolysis of triphosphoric and pyrophosphoric acids ; reaction teinpcratures of 40 and 30' were employed, and a t each temperature hydrolysis rates were determined a t two levels of acidity. N o additional inert salt was present in these rate runs beyond that introduced in the sample preparation procedure. The stoichiometric hydrolysis reaction C:,IlyOj-CM?OPOsH,
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phosphate under equivalent conditions will proceed largely by cleavage a t the terminal phosphate grouping, without a significant contribution from slower hydrolysis a t the internal sugar-phosphorus bond. In this connection, it is also of considerable interest that both the acid-catalyzed4*6and enzymatically-catalyzed7 hydrolysis of the related polyphosphate, triphosphoric acid, involve cleavage of a terminal phosphate residue. Further, the data of Table I permit a rough comparison of the temperature coefficients for the hydrolysis of FPA a t relatively low (0.025 N ) and high (0.09 Nj levels of added acid. For the temperature interval employed, both low acidity (runs 1 and 3) and high acidity (runs 2 and 4) determinations lead to virtually the same temperature coeficient for kl,i.e., a factor of about 3.7 for 10.3'. This would imply that both the catalyzed and uticatalyzed contributions to observed kl values have the same temperature dependence. As a final observation, the results of run 5 employing HC1 as catalyst (compared to 1 and 2 using IIzS04) point to the relative insensitivity of the hydrolysis to the nature of the strong acid used as catalyst. Experimental
The barium salt of fructose-&phosphoric acid was used was followed by colorimetric evaluation of inorganic direct!y as supplied by the Nutritional Biochemicals Corphosphate evolved, using the method of Lowry poration, Analyses for total hydrolyzable phosphate and barium ion indicated a maximum purity of 95.5% with and Lopeze5 First-order rate constants were for respect to barium fructose phosphate. Triply distilled calculated for the exceedingly slow hydrolyses over water was used in all rate determinations. roughly the first several per cent. of each reaction, In a given rate run, a weighed portion of barium salt was corresponding to a total reaction time of about 10 dissolved completely in about 25 ml. of water, giving a pale solution, To this was added the equivalent quandays. Values for these constants and their ob- yellow tity of sodium sulfate, in small portions with continual served deviations are sumniarized in Tablc I . stirring. The required amount of a standard solution of acid catalyst was then added by pipet, with stirring. After 'LABLE I standing in the cold for about five minutes, the mixture was directly into the 50-ml. volumetric flask used as the L ~ C I D - C ~ Y ~ A L Y Z EHYDROLY-SIS D OF F R U c . l - O S ~ - 6 - P ~ i u S P ~ ~ o R Ifiltered c rcaction vessel, with about 13 i d . of water being used in the Ac1u (FP.1) transfer and subsequent washing. The solution was then Reactiun placed in the constant temperature bath, allowed t o come temperaturc,'l Initial concentrations hi, to temperature, and made to volume with preheated water. Run oc. Acid catalyst, S FP.4, .\I sec.-I X 10' Aliquots (2 ml.) of reaction mixture were withdrawn a t 1 38.93 H2S04, 0.0247 0,0210 1 , 0 9 i.0 . 06 intervals of roughly 24 hours, delivered into about 40 ml. of ,0210 1 . 7 3 i .07 water containing 0.4 millimole of sodium acetate, made to 50 HzSOd, ,0914 2 39.!13 .0210 4 . 11 i .10 nil., and finally analyzed colorimetrically6 for inorganic phosH2S04, ,0247 ;; 50.33 phate. Concentrations of FPA were calculated from initial .(I210 6 . 5 2 f . 4 i values and the subsequent analytical values for phosphate. HzS04, .(1
