SPECIFICITY IN CHOLINESTERASE REACTIONS 177 



Assuming unit negative charge for the anionic enzymic site and equating 

 the extra free energy of binding of the cationic member of each pair to the 

 potential energy we may calculate R. 



The effective dielectric constant is calculated to be approximately 20. 



I would like to mention, as Dr. Pressman mentioned earlier, that there 

 is an approach to the closest contact possible for a quaternary methylated 

 ammonium ion and some negative grouping, because the quaternary structure 

 has about 3.5 Angstrom radius and, if we take one and a half A for the other 

 group — oxygen or whatever it may be — that bears the negative charge, the 

 closest approach is 5 A. 



There are several other ways of checking ionic binding, but I would just like 

 to briefly mention one other. We can change the ionic strength of the medium 

 and that is a useful procedure because the expected variation is independent of 

 the distance of approach, except in a correction term. It has been found that the 

 iMichaelis-Menten constant which we will temporarily assume is the enzyme- 

 substrate dissociation constant, decreases quite rapidly as the ionic strength 

 decreases. Again assuming that the anionic site charge is one electronic unit, 

 the effective dielectric constant comes out to about 25. 



So one can make the story iit with a single ionic charge for the anionic site. 

 It does not have to be so; it might be the summation of many charges at dif- 

 ferent distances with different effective dielectric constants, but at any rate, 

 the data fit a single charge satisfactorily, and there is little doubt that there 

 is an anionic site. 



There is one other type of experiment which I should like to mention, which 

 pertains to this anionic site story. So far we have compared charged and un- 

 charged inhibitors. This can also be done with substrates. Whitaker and Adams 

 compared acetylcholine with its carbon analogue, 3,3 dimethyl butyl acetate 

 (Adams and Whitaker, 1950). We have compared acetylcholine with its tertiary 

 analogue, dimethylaminoethyl acetate, as a function of pH (Wilson and Berg- 

 mann, 1950a). The pKa of this compound is 8.3, so that by going to the acid 

 side or the alkaline side we can make a positively charged or an uncharged 

 substrate. In Fig. 2 the velocity is given as a function of pH. Just as one might 

 anticipate, a curve is obtained that drops between pH 8 and 9, indicating 

 that the cationic species is much more readily hydrolyzed. 



So we have, I think, established rather well that there are ionic forces of 

 attraction between the substrate and the enzyme which play an important role 

 in the binding and catalysis. 



Next one might ask whether there are binding forces associated with the 

 methyl groups of the cationic head. It might be suspected that there would be 

 van der Waals forces of attraction between these groups and hydrocarbon 

 portions of the protein. 



Rather than go into this too far, I might just explain that one can take any 



