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Above about 45°C, it is observed that the reaction rate increases less 

 rapidly and then decreases as the temperature is raised. If the trypsin 

 is cooled, it regains its old activity. This behavior may be represented 

 as a competitive reaction 



E ^ E x ^ E' 



where £* is an activated form and E' a reversibly denatured form of the 

 enzyme. 



At still higher temperatures, above 70°C, the reaction rate decreases 

 further. On cooling, the enzyme does not regain its original activity. 

 Under these conditions, the trypsin is said to be irreversibly denatured. 

 In the symbolism of absolute rate theory, this situation can be repre- 

 sented by 



E' ^ E' % -s. E" 



where E" is the irreversibly denatured form. 



It was noted previously that a monomolecular reaction such as a 

 denaturation is not diffusion limited. This in no way implies that the 

 reaction does not depend on collisions. Quite the opposite is the case. 

 In the kinetic theory, heat energy is represented as random motion of the 

 constituent particles. In a fluid, the random vibrations of the protein 

 molecule are thought of as continually bringing about collisions between 

 one part of the protein and another, and between the parts of the protein 

 molecule and the vibrating fluid molecules. Denaturation will occur 

 when an excess of energy is delivered by a series of collisions to a particu- 

 lar bond or group of bonds. 



Absolute rate theory can be used successfully to describe denaturation, 

 but collision theory has little to offer. The difficulties of applying 

 collision theory to a denaturation-type reaction are worse than for a 

 bimolecular reaction. Not only is one unable to quantify collision 

 frequencies and probabilities, but one does not even know the location 

 or the type of the critical colliding groups. Thus, except for implying 

 (correctly) that an Arrhenius coefficient should exist, collision theory 

 per se can yield little insight into the nature of denaturation. It offers 

 no explanation for the spread of values of the energy of activation for 

 denaturation, although it does imply correctly that these should be high 

 in order that the enzymes be stable at room temperature. 



The proponents of absolute rate theory have emphasized its applica- 

 tion to denaturation studies. On purely theoretical grounds, there is 

 less reason in hesitating to apply it to these reactions than to any others. 

 The reactions are essentially monomolecular, hence, there is no problem 

 of diffusion. The equilibrium between the activated form and the 



