412 Thermodynamics of Enzyme Reactions /22 : 4 



In addition to the foregoing, in the derivation of the absolute rate of 

 transformation of the activated complex to its products, there are a 

 number of approximations which could easily not apply to enzyme 

 reactions. Unless one is able to observe the activated complex by 

 physical measurements or can verify the entropy of activation by an 

 independent set of measurements, one cannot rule out the possibility 

 that absolute rate theory does not apply to some (or even all) enzyme 

 reactions. In spite of these uncertainties, absolute rate theory forms 

 the basis in terms of which reaction-rate data are often presented. 



4. Denaturation Studies 



Absolute rate theory applies in liquids only to reactions which are not 

 diffusion limited. 3 All reactions which involve changes in one molecule 

 only are by definition not limited by diffusion. This section deals with 

 a particular type of monomolecular reaction characteristic of proteins. 

 The changes produced by these reactions are called denaturations ; they 

 result from submitting the protein molecules to various physical changes 

 such as heat, cold, vibration, and so on. If denaturation does not 

 proceed too far, it may be reversible; under more extreme conditions, 

 denaturation becomes irreversible. 



Enzymes are particularly suitable for denaturation studies because 

 small changes in their internal structure may produce dramatic changes 

 in their rates of reactivity. Almost all enzymes rapidly lose their 

 activity irreversibly at temperatures around the boiling point of water. 

 By and large, the rate of denaturation depends exponentially on the 

 reciprocal of the absolute temperature. The Arrhenius coefficients (or 

 activation energies) vary widely but are all larger than 15 kcal/mole. 



A typical denaturation study involves the rate at which trypsin 

 digests a given protein. To observe reversible denaturation, one 

 measures the rate of reaction at various temperatures. In the low 

 temperature range, the slope of the Arrhenius plot is about 10 kcal/mole 

 and the reaction can be described by the typical equations 



E + S^E-S 



E-S-^E + Products 



where E is trypsin, S is the protein, and the products are the hydrolyzed 

 (that is, split) protein. 



3 If the reaction is diffusion limited, absolute rate theory may be applied to 

 the diffusion, but not to the reaction itself. 



