KARL F. GUTHE 77 



7 



but the rate always increases with temperature slightly less rapidly than 

 required by the Arrhenius i-elation, as expected when denaturation is a 

 significantly fate-limiting factor. 



The effects of pressures at different temperatures are also consistent 

 with reversible denaturation. Pressure increases the activity at high tem- 

 peratures and decreases it or leaves it unchanged at lower temperatures 

 (fig. 3, right). This again suggests that two different processes are af- 

 fected. If reversible denaturation increases as the temperature increases, 

 most of the enzymatic sites are native at low temperatures. Inhibition 

 at low temperature is then again a direct effect on the rate process, while 

 pressure renatures enough denatured sites at higher temperatures so that 

 the over-all activity is increased. 



With the previous values for pK and for the volume changes in the 

 rate process and in reversible denaturation, the temperature data indicate 

 that the heat of reversible denaturation is 12,600 calories per mole and 

 the heat of activation is 31,000 cal/mole for the breakdown of the enzyme- 

 substrate complex. These values were calculated on the assumption that 

 neither volume change is temperature-sensitive. Preliminary experiments 

 show that pressure inhibition at pH 6.3 decreases as the temperature de- 

 creases, which indicates that the volume of activation for the rate process, 

 at least, decreases with temperature, like the corresponding volume change 

 in bioluminescence (7). When the temperature-dependence of the volume 

 changes are more accurately determined, the heats will require correction. 



PRESSURE 



It remains to be shown that the pressure curve is also consistent with 

 reversible denaturation. If activity is plotted against pressure, different 

 experiments show different amounts of increase. A pressure of 12,000 

 pounds per square inch increases the activity 2^/2 to 3-fold. The reason for 

 this variation is not clear. Some of it may result from small changes in 

 pH or in other constituents of the solution, such as potassium ion concen- 

 tration. At present, we are especially interested in the shape of the activity: 

 pressure curve. The different experiments coincide if the increase at each 

 pressure is plotted as percentage of the increase at a pressure of 12,000 

 pounds/in-. In figure 4, the value of Vp/v at this pressure was arbitrarily 

 set at 3.0, the observed value in most experiments, and other experiments 

 were corrected on the percentage basis just described. 



The data in figure 4 are plotted on a logarithmic scale. If pressure af- 

 fected only the rate of reaction, the points would be linear, and the volume 

 of activation could be calculated from the slope. At the lower pressures, 

 the slope corresponds to a volume of activation of —40 cc/mole. Above 



