Joyce F. Miller
and Joseph G. Cory University of South Florida Tampa, Flor~da33620
II
Activation Energies for a Base-catalyzed and Enzyme-Catalyzed Reaction
In discussion of the effects of catalysts on reaction rates, it is always pointed out that the effect of a catalyst (e.g., metal ion, enzyme) on a given reaction is to lower the Arrhenius activation energy. The catalyst does not influence the equilibrium constant of the reaction. An experiment, suitable for either biochemistry or physical chemistry laboratory, has been devised to demonstrate the difference in Arrhenius activation energies between an enzyme-catalyzed and basecatalyzed reaction. The reaction studied was the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate. p-Nitrophenyl phosphate is an excellent substrate for alkaline phosphatase and is easily hydrolyzed in basic solutions. The rate of hydrolysis of this compound can be followed spectrophotometrically by the absorption at 410 nm of the p-nitrophenolate ion.' The rates of hydrolysis for the enzyme-catalyzed and base-catalyzed reactions were measured under identical conditions of substrate concentration (5 X M), and Tris buffer (2-amino-2-hydroxymethyl-1,3-propanediol, 0.1 M , adjusted to pH 9.2). The reaction rates were measured at different ranges of temperature depending on the catalyst used. The rates of the enzyme-catalyzed hydrolysis were measured over the temperature range 0-50°C, whereas the rates for the base-catalyzed hydrolysis were measured over the range 6&80°C. The natural logarithm of the rate of hydrolysis is plotted against the reciprocal of the absolute temperature and the Arrhenius activation energy is calculated from the slope of the line (In v = -E,/RT constant).
+
Experimental
The absorbance of the p-nitrpphenolate ion at 410 nm follows Beer's Law (the molar extinction coefficient for the p-nitrophenolate ion under these conditions is 2.1 x lo4). The rates of the base-catalyeed hydrolysis were measured a t 60", 65", 70a, 75', and 80°C. To avoid errors due to temperature equilibration, six samples of the Tris-buffer 0.1 M, pH 9.2, (2.7 ml) were equilibrated for 10 min at the designated temperature. The substrate, p-nitrophenyl phosphate, 0.05 34, (0.3 ml) was then added to the tubes. One sample was immediately removed and placed in an ice-bath and this sample served as the t = 0 sample. The other samples were removed st regular time intervals and also cooled in an ice bath. Since the rate of hydrolysis changed markedly a t the various temperatures, the time-interval for removal of the various samples varied with the temperature. Samples were removed st 20, 15, 10, 5, and 5 min for the experi1 GAREN,A,, AND LEYINTHAL, C., Biochim. Biophys. Ado, 38, 470 (1960). Alkaline phosphatase, calf intestinal, Type I, purchased from Sigma Chemical Company is a. suitable preparation for these studies.
,,
,
.. ,-
Temperature dependence of the velocity of p-nitrophenyl phosphate hydrolysis. Tho dota for the hydrolysis of p-nitrophenyl phosphate b y barecotolysi$ are shown b y the dosed circler, while the dota for the enzyme satolyzed reaction are shown by the closed rquorer.
ments at 60°, 65", 70", 75' and 809C, respectively. The rates of the enzyme-catalyzed hydrolysis were measured s t 0°, loD,20°, 28", 38', and 50% Seven samples (1 ml aliquots) of a solution of p-nitrophen~lphosphate (0.005 moles dissolved in 1 l of 0.1 M Tris-buffer, pH 9.2) were equilibrated for 5 min prior to the addition of 0.1 ml of alkaline phosphatase2 (approx. 1 mg/50 ml, the amount of eneyme added depending on the source and purity of the enzyme). The enzyme-catalyzed hydrolysis was stopped by the addition of 7.0 ml of 0.03 N NaOH to the sample tubes at 6 m i n intervals from the time of addition of the enzyme. The rstes of hydrolysis were determined from the slope of a graph of pmoles of p-nitrophenol (or absorbance a t 410 nm) against time.
Results
After the rates of the hydrolysis of p-nitrophenyl phosphate in the base-catalyzed and enzyme-catalyzed reactions have been calculated for each temperature, plots of in v against 1/T are made from these data. The Arrhenius activation energy, E., is calculated from the slope of the resulting line for each case [E, = -(slope) (R)]. Results from the experiments described above are shown in the figure. From the slopes of these curves, an E, of 30 kcal/mole is calculated for the base-catalyzed reaction, while an E, of 7.8 kcal/mole is calculated for the enzyme-catalyzed reaction. It is seen in the curve for the enzyme-catalyzed reaction, that the rates of hydrolysis fall off at the two highest temperatures due to the denaturation of the enzyme a t these temperatures. These points are ignored when the line is drawn through the other points. The large differencein E i s for the base-catalyzed and enzyme-catalyzed reactions make an impressive display of the effect of catalysts on the Arrhenius activation energy. Since the E, is calculated from the slope of the line, it does not matter whether the rates of reactions are plotted in terms of &moles of product/time or in terms of absorbance/time. Volume 48, Number
7,July 1971
/
475
