INTERACTION WITH CHOLINESTERASE 285 



cationic group of the substrate. The evidence lies mainly in the different 

 bindings of ionic and nonionic substrates and inhibitors to the enzyme, and 

 in the effects of pH and ionic strength on the binding. There is some dis- 

 agreement as to whether there are one or two anionic groups on the true 

 cholinesterase: Wilson (1957) believes that the results are compatible with 

 a single anionic site while Bergmann (1958) has brought forth reasons for 

 assuming that two such groups interact with a single cationic substrate or 

 inhibitor. Before discussing this problem further, let us consider the results 

 obtained. 



Three general methods have been used to estimate the magnitude of the 

 ion-ion interaction: (1) the comparison of cationic substrates or inhibitors 

 with the uncharged analogs, (2) the determination of the difference in 

 binding at two pH's sufficiently apart so that the substrate is charged at 

 one pH and uncharged at the other, and (3) the comparison of inhibition 

 of true and pseudocholinesterase by a cationic inhibitor, inasmuch as the 

 latter enzyme does not have an anionic group (or has one less than the true 

 cholinesterase). The results of such study are summarized in Table 6-22 

 and calculations of the equilibrium interaction distance are given for as- 

 sumptions of either one or two anionic sites. The average charge separation, 

 assuming one anionic group at the active site, is 5.55 A; this would imply 

 an approximate distance of 6.15 A between the N+ and the carbon atom 

 of the carboxylate group, which is very close to the separation at contact 

 of the van der Waals' surfaces. If two anionic groups are assumed on true 

 cholinesterase, the calculated separation of 7.18 + 0.6 = 7.78 A would 

 not allow the interposition of a water molecule and it must be concluded 

 that the data in Table 6-22 point to a single anionic group. The pseudo- 

 cholinesterase would then possess no anionic group immediately adjacent 

 to the interacting cationic group. The calculations of dg from the K^ of 

 tetramethylammonium ion, obtained by extrapolation from higher analogs, 

 made by Bergmann (1958) are probably not valid since no account was 

 taken of interactions other than ion-ion. Bergmann stated that van der 

 Waals' forces cannot make an important contribution to these inter- 

 actions, but this is certainly incorrect, especially when the displacement of 

 water from the groups is considered. 



Interactions of Alkylammonium Ions with Cholinesterase 



The possibility of determining the dispersion contribution to the inter- 

 action energy of the — N(CH3)3^ end of the acetylcholine molecule led 

 Wilson (1952 a) to examine the relative inhibitory potencies of variously 

 substituted ammonium ions. The results are shown in Table 6-23. Removal 

 of one methyl group from the tetramethyl compounds does not reduce the 

 binding, which indicates that the entire group does not fit into a cavity 

 but that one methyl group is directed away from the surface. Removal of 



