284 6. INTERACTIONS OF INHIBITORS WITH ENZYMES 



cp— ■^N(CH3)3 but not with CH3 — "^N(CH3)3, the difference in binding energy 

 being 4.7 kcal/mole (Pressman et at., 1946). Induction forces involving the 

 benzene ring are of primary importance in the binding of these haptens. 



Effect of Hydration on Hapten-Antibody Binding 



We noted previously that the free energy for binding of the divalent 

 hapten "HOgAs-cp-NHCO-cp-CONH-cp-AsOgH" to antibody for 

 p-azophenylarsonate is mainly derived from the entropy change due to 

 water displacement, and such must occur to a greater or lesser extent in 

 all combinations of hapten and antibody. However, when water is bound 

 tightly to a group on the hapten, so that it is not displaced upon interaction, 

 it alters the contour of the molecule much as a substituent group would 

 do. This is clearly seen when a pyridine ring replaces the benzene ring of 

 haptens reacting with antibody to the azobenzoates (Pressman and Siegel, 

 1957). The pyridine carboxylates, for example, are bound with 1.2-1.5 

 kcal/mole less energy than benzoate to the antibody for o-azobenzoate. 

 These effects are greater than occur upon substitution of a chlorine atom 

 on the benzene ring, indicating that the pyridine nitrogen atom has a 

 greater effective size than the chlorine atom, which could result only 

 from the hydration of the pyridine. It is known that pyridine is hydrated in 

 aqueous solution from the fact that it is miscible with water whereas ben- 

 zene is only slightly soluble; the heat of hydration of pyridine is 12 kcal/ 

 mole. Thus one cannot always consider just the contour of the hapten mol- 

 ecule itself but the spatial modifications produced by hydration. 



INTERACTION OF SUBSTRATES AND INHIBITORS WITH 



CHOLINESTERASE 



The quantitative approach to the treatment of hapten-antibody inter- 

 actions was presented by Pauling in lectures at Oxford in 1948. He pointed 

 out that this method was applicable to enzymes and Adams and Whit- 

 taker published from Oxford in 1950 the original treatment of enzyme 

 complexes in terms of intermolecular forces. The enzyme cholinesterase 

 was convenient to use for this purpose and since that time several investi- 

 gations have been directed toward the elucidation of the active site and 

 the forces it exerts on both substrates and inhibitors. 



Electrostatic Interaction of Ionic Groups 



The true erythrocyte cholinesterase, which splits acetylcholine and other 

 choline esters at a very rapid rate, would appear to possess within its active 

 site an anionic group, presumably a carboxylate ion, which reacts with the 



