SPECIFICITY IN CHOLINESTERASE REACTIONS 179 



are more or less spherical and one group must stick out into the solution unless 

 the protein wraps itself around this group. If we assume that the protein does 

 not undertake such an envelopment, then the group must stick out into the 

 solution and be without binding properties. In that case, we would expect 

 that it would make no difference whether there were four or three. However, 

 as we further decrease the number of groups, binding properties are lost. 



Obviously, there are going to be questions of hydration and dehydration 

 but, neglecting them for the moment — they have been considered by others — ■ 

 we can derive an order of magnitude here. A factor of 7 is worth about 1.2 kilo 

 calories/mole binding energy and when we can compare that with the latent en- 

 ergy of evaporation of methane, which is about 2 kilocalories/mole, we come 

 out with about the right order of magnitude for substituting a methyl group 

 for hydrogen. 



I do not want to go into this question further, but prefer to discuss what 

 other sources of binding could be involved. 



Another important feature is the carbonyl group. Chemists in the early 

 days used to write this group with a negative and a positive charge, but now 

 it is considered to be a resonance structure in which this form contributes 

 about equally with a pure double bond. If we leave it in this dipolar form, we 

 have a carbon atom which is looking for electrons. We believe a weak covalent 

 bond is formed between this atom and some basic group in the active site of 

 the enzyme. 



We have used a number of series to investigate this prospect, but I will give 

 the data for only one series. That series is ethyl acetate, ethylchloroacetate 

 and acetic anhydride, which are presented in Table 3 with the INIichaelis- 

 Menten constants (Wilson, 1952). I might just mention that, of course, it is 

 not surprising that acetic anhydride should be hydrolyzed by this enzyme. 

 This compound is not much different from an ester. But the organic chemist 

 will right away know that in going down this series he is making a tremendous 

 increase in the electron-seeking properties of the carbonyl carbon atom, in the 

 electrophilic properties. The ]Michaelis-]\Ienten constants also decrease rapidly. 

 These changes correspond to from 3 to 5 kilocalories and I think the only way 

 one can account for this large difference in binding is to assume that in fact 



TABLE III 



Substrate 



Ethyl acetate 



Ethyl chloroacetate . 

 Acetic anhydride. . . 



5 X 10-1 



6 X 10-3 

 2 X 10-4 



Relative h* 



12 

 13 

 13 



* 



Acetylcholine = 100. 



