Hydrophobic areas on the active surface of cholinesterases.

A. The "topography" of the hydrophobic areas The analysis of all the results described above allows us to form a general idea about the localization of the hydrophobic areas on the active surface of butyrylcholinesterase and acetylcholinesterase and about some of the peculiarities of these areas (6, 31, 52). A diagram of the localization of the hydrophobic areas on the active surface of cholinesterases is given in figure 13. In the region of the anionic site there are two hydrophobic areas: A1 directly surrounding the anionic group and A2 located at some distance from the anionic group, "behind" it, outside of the true anionic site. Probably area A1 plays a more important role in butyrylcholinesterase than in acetylcholinesterase. Area A2 is not equal in the different enzymes: in butyrylcholinesterase its length corresponds to 6-carbon chain and in acetylcholinesterase it can accommodate an 8-carbon chain. The spatial configuration of this area makes it complementary to the radicals containing the highly branched tertiary butyl group separated by 4 to 6 methylene groups from the thiol sulfur atom. The scheme supposes that in the vicinity of the esteratic site of butyryicholinesterase there are two hydrophobic areas (E1 and E2) separated by a hydrophilic group. The total length of this hydrophobic area corresponds to a 7-carbon chain. Both parts of this hydrophobic area (E1 and E2) seem to be complementary to the radicals of straight-chain structure, but the first part (A1) is not so strictly limited and branched hydrocarbon radicals can also be sorbed onto it. In the vicinity of the esteratic site of acetylcholinesterase there is probably a single hydrophobic area whose structure differs considerably from that of the area E1 in butyryicholinesterase: the area in acetylcholinesterase is strictly complementary to the isohexyl radical. Neither the area E1 in butyryicholinesterase nor the area E in acetylcholinesterase is suitable for the sorption of radicals containing a tertiary butyl group. Thus marked peculiarities in the structure and length of different hydrophobic patches on the active surface of butyryicholinesterase and acetylcholinesterase have been established. It seems very probable that these peculiarities are responsible for the different properties of the two cholinesterases. B. The possible biological significance of the hydrophobic areas on the active surface of cholinesterases and cholinoreceptors It is natural to ask what is the biological purpose of the structure described above on the active surface of cholinesterase. It is relatively easy to explain the purpose of the hydrophobic area E1 immediately surrounding the anionic group: it is adapted to interaction with the methyl groups attached to the nitrogen atom of acetylcholine. But what is the purpose of the spacious hydrophobic regions A2 and E (E1 and especially E2), which are situated beyond the catalytic center of the enzyme? It does not seem likely that these hydrophobic areas were formed in the course of evolution only as accidental details of the enzyme structure. The following assumption seems more probable (52). The acetylcholine molecule, containing a charged nitrogen group and a carbonyl group, is very hydrophilic. It can be sorbed not only at the active center but at many other polar groups of the polypeptide chains of the enzyme. Such a "parasitic" sorption would decelerate the interaction of the acetylcholine molecule with the active center. The extensive hydrophobic areas (A2, E1, and E2) interfere with such a parasitic sorption. The hydrophilic acetylcholine molecules are "thrown out" of these hydrophobic areas and the probability of their coffision with the active center of the enzyme increases. One could say that these hydrophobic regions constitute energetic "hills" from which the acetylcholine molecule is rolled down to the active center. Thus the "effective section" of the enzyme is actually enlarged, and this increases the rate of hydrolysis of acetylcholine. In this connection another problem, that of the structure of the active center of the cholinoreceptor, must be touched upon. There are serious reasons to suppose that hydrophobic areas are present around the active center of the cholinoreceptor (53, 66). The existence of a hydrophobic area adapted to the interaction with the methyl groups of the cationic head of the acetylcholine molecule was recently postulated by Barlow (14). This area directly surrounding the anionic group of the receptor is probably similar to area E1 in cholinesterase. In the region of the so called "esterophilic site" (53) of the receptor a small hydrophobic area adapted to the interaction with the methyl group attached to the carbonyl carbon of acetylcholine also has been postulated (14). Belleau assigned an important role to hydrophobic interactions in the reactions of acetylcholine with the active centers of both cholinesterases and cholinoreceptors (17, 18). But, in addition, some other, more spacious hydrophobic areas must also exist close to the active center of the receptor. This idea is supported by the enormous amount of experimental data that has accumulated in the field of structure-activity relationships for the cholinolytic agents. Long ago Bovet and Bovet-Nitti (22) formulated the empirical rule that increasing the molecular weight of the aeetylcholine molecule by attaching hydrocarbon groups to its acidic and alcoholic ends converts it into a cholinolytic agent. (They called this process "allourdissement" of the acetylcholine molecule.) Actually it is well known that the introduction of hydrophobic radicals, such as phenyl, cyclohexyl, or large aliphatic radicals, in the acidic part of the acetylcholine molecule gives potent blocking drugs. With gradual "allourdissement" of the acetylcholine molecule its cholinomimetic activity gradually diminishes and the cholinolytic properties become more and more pronounced until a certain limit is reached. The replacement of methyl groups attached to the nitrogen atom by ethyl groups has a similar effect (14, 50, 66). This effect seems quite comprehensible. For the cholinomimetic action not only rapid sorption, but also rapid desorption of the drug from the receptor is needed. The deceleration of the desorption leads to a blocking effect (71). The introduction of heavy hydrophobic groups in the acetylcholine molecule enhances the interaction with the hydrophobic areas situated outside the active center of the receptor and hinders the desorption. It is natural to suppose that in the case of the action of acetylcholine itself the biological function of such hydrophobic areas in the vicinity of the active center of the receptor is the same as with cholinesterase: it enlarges the "effective area" of the active center of the receptor and increases the probability of an effective collision of the acetylcholine molecule with the receptor surface.