SPECiriCITY IN CHOLINESTERASE REACTIONS 175 



one should look for what we have called an anionic site in the protein molecule; 

 some negative site which by ionic forces helps to bind this group. The role of 

 ionic forces in hapten-antibody reactions has been studied by Pressman and 

 Pauling and presented in this volume. With enzymes, this question can be 

 investigated in several ways. 



One method we have used a good deal (Wilson and Bergmann, 1950a ; Berg- 

 mann, Wilson and Nachmansohn, 1950), has been with competitive inhibitors. 

 These are inhibitors which produce an inhibition which decreases with increas- 

 ing substrate concentration, so that one can interpret that they are competing 

 for the same site. It is often apparent, however, just from their structure that 

 they are competing for the same site. Prostigmine, 



O 



-O— C— N(CH3)2 



N(CH3); 



for example, clearly resembles acetylcholine. 



The tertiary compound eserine is related to prostigmine. Both are very good 

 inhibitors of cholinesterase, effective at about 10~^ M concentration. The per- 

 tinent difference between these inhibitors is that while prostigmine is a quater- 

 nary ion and is therefore always positively charged no matter what the pH, 

 eserine is a tertiary compound and is therefore positively charged in acid 

 solution but uncharged in alkaline solution. 



By acid or alkaline I mean with respect to its pKa, which is about 8.5. 



Fig. 1 shows the inhibition of these compounds as a function of the pH. 

 Inhibition by prostigmine (black circles) does not vary with pH. Now, that is 

 of importance in our interpretation because it shows that within this range of pH, 

 those changes that may occur in the protein do not affect the inhibition. 



However, eserine, which at pH less than 8.5 is mainly cationic and at pH 

 greater than 8.5 is mainly neutral, shows a very marked change; the inhibition 

 decreases in alkali (A)- So, we can measure the binding constants for the un- 

 charged and charged forms. 



There are other ways we can make similar measurements. For example — 

 nicotinamide which is uncharged in neutral solution (nicotinamide is also an 

 inhibitor for this enzyme) can be compared to its N methyl derivative, which, 

 of course, is a quaternary ion, and is positively charged. 



And, to take still another case, one can compare carbon and nitrogen ana- 

 logues as inhibitors (as was done by Whitaker and Adams for substrates). For 

 example, isoamylalcohol can be compared with dimethyl amino ethanol. The 

 latter compound is cationic at neutral pH. 



