126 O. MEYERHOF, R. SHATAS, A. KAPLAN VOL. 12 (1953) 



The procedure of measurement was described in an earlier paper^^. The concentration 

 of nucleic acids in the heat assay was less than M/5000. At the end of the measurement, 

 the reaction was stopped by addition of 4 ml of 40% trichloroacetic acid to the reaction 

 mixture and aliquots taken out for the determination of orthophosphate formed. No 

 measurable heat was evolved when the enzyme was blown in, in the absence of 

 substrate. 



The value of measured heat effect given in Table VI refers to all three P-O-P bonds of trimeta- 

 phosphate. A rough estimate of the heat of neutralization yields 700 cal/mole*. After subtracting this 

 figure from the measured heat effect, we find AH = — 18,600 cal/mole as an approximate value for 

 the change in enthalpy. Thus, the hydrolysis of each P-O-P bond of trimetaphosphate is accompanied 

 by a decrease in enthalpy of approximately — 6,000 cal/mole at a neutral pH. Our value is two times 

 higher than the result listed with an older measurement of acid hydrolysis of an unidentified meta- 

 phosphate by Giran^". 



DISCUSSION 



The knowledge of the change in enthalpy does not directly answer the question 

 concerning the change in free energy; however, its magnitude can be evaluated if some 

 additional data are available. In a previous paper we estimated the entropy factor, TAS, 

 of pyrophosphate hydrolysis at 1,000 cal/mole^''. When trimetaphosphate is hydrolyzed, 

 we may expect three times as large an increase in entropy because three P-O-P bonds 

 are broken. Secondly, according to the Boltzmann's relation between probability and 

 entropy, breaking down the trimetaphosphate contributes an additional increase in 

 enthalpy because the formation of its ring structure is less probable than that of the 

 single P-O-P bond of pyrophosphate. In the third place, even if we assume that at pH 7 

 the trimetaphosphoric acid is completely dissociated, an increase in acidity with the 

 formation of orthophosphate would still occur, thus decreasing the over-all change in 

 free energy. These reasons enable us to estimate the decrease in free energy accompanying 

 the hydrolysis of trimetaphosphate at not less than 21,000 cal/mole. Thus, the P-O-P 

 bond of trimetaphosphate should be regarded as a "high-energy" bond of intermediate 

 range. It is very likely that other metaphosphates have similar bond energies. However, a 

 definite proof can be supplied only by suitable measurements. 



SUMMARY 



An enzyme extract of yeast has been prepared which hydrolyzed inorganic trimetaphosphate 

 to orthophosphate. The highest rate of enzymic hydrolysis has been observed at pH 7. Among 

 divalent kations magnesium and manganese have been found to be the best activators. Fluoride, 

 cyanide, and, to a lesser degree, azide inhibited the enzyme. By the addition of glycerol the activity of 

 the enzyme at 33° C was much better preserved as compared to a water solution. The heat effect of 

 enzymic hydrolysis of trimetaphosphate, 19,300 ^ 900 cal/mole, was measured with the isothermal 

 constant-flow calorimeter of Ohlmeyer; an estimate of the decrease in enthalpy yielded 18,600 

 cal/mole for the ionization states of reactants and products at pH 7 and 33° C; likewise, the decrease 

 in free energy was estimated at more than 21,000 cal/mole. Thus, the "high energy" content of the 

 P-O-P bond of trimetaphosphate has been demonstrated. 



RfiSUMfi 



Nous avons prepare un cxtrait des enzymes de levure capable de former I'orlhophosphate en 

 hydrolysant le trimetaphosphate inorganiquc. Le maximum de I'activite de I'enzymc a ete observe 

 avec un pH de 7. Nous avons obtcnu I'activation la plus forte de I'enzymc par le Mg et le Mn de tous 



* Assuming a complete dissociation of trimetaphosphoric acid at a neutral pH, the heat of 



neutralization of the partial second dissociation of orthophosphoric acid (p^2 — ^•^) ^*^ P^^ 7 '^ 



[H+l 



3 X =r- X 380 or 700 cal per mole of trimetaphosphoric acid hydrolysed. 



[H+] + Ag 



References p. I2y. 



