Serine-type acetylcholinesterase

Over 80 different (3-lactamases are now known. One classification is a system that divides the enzymes into three classes A, B, and C. Classes A and C are active-site serine enzymes. The serine residue in class A enzymes is at position 70. This class contains four major (3-lactamases 749/C (from B. licheniformis), PCI (from S. aureus), 569/H P-lactamase I (from B. cereus), and PBR322 and RTEM (from E. coli). As with other serine-type hydrolytic enzymes (acetylcholinesterase, trypsin), the mechanism of action requires initial formation of an acylated enzyme, in this case acylation of ser-70 followed by hydrolysis of the derivative to regenerate the enzyme ... 

Malathion and parathion contain a P=S grouping, exemplifying a further carbonyl analogue, in which phosphorus replaces carbon, and sulfur replaces oxygen. Nevertheless, the same type of chemistry occurs, in which the serine hydroxyl of the insect s acetylcholinesterase attacks this P=S electrophile, followed by expulsion of the leaving group, here a thiolate. The esterified enzyme, however, is not hydrolysed back to the original form of the... 

It is important to know that the inhibition of acetylcholinesterase by OPs is through an attack on the relatively positive phosphorus atom by the hydroxyl group of a serine residue at the enzyme s site of action. Electron withdrawing substitutions within the OP tend to make the phosphorus more positive and, therefore, more reactive. Unfortunately, this type of substitution also makes the compound less stable hydrolytically. The discovery and development of OP insecticides has always been a balance between activity against the enzyme of the insect, selectivity in comparison with mammalian systems and stability within the insect. The binding of OPs to acetylcholinesterase is often irreversible. Typical OP insecticides are shown in Figure 3.3. 

The previous discussion of amino acid catabolic disorders indicates that catabolic processes are just as important for the proper functioning of cells and organisms as are anabolic processes. This is no less true for molecules that act as neurotransmitters. To maintain precise information transfer, neurotransmitters are usually quickly degraded or removed from the synaptic cleft. An extreme example of enzyme inhibition illustrates the importance of neurotransmitter degradation. Recall that acetylcholine is the neurotransmitter that initiates muscle contraction. Shortly afterwards, the action of acetylcholine is terminated by the enzyme acetylcholinesterase. (Acetylcholine must be destroyed rapidly so that muscle can relax before the next contraction.) Acetylcholinesterase is a serine esterase that hydrolyzes acetylcholine to acetate and choline. Serine esterases have catalytic mechanisms similar to those of the serine proteases (Section 6.4). Both types of enzymes are irreversibly inhibited by DFP (diisopropylfluorophosphate). Exposure to DFP causes muscle paralysis because acetylcholinesterase is irreversibly inhibited. With each nerve impulse, more acetylcholine molecules enter the neuromuscular synaptic cleft. The accumulating acetylcholine molecules repetitively bind to acetylcholine receptors. The overstimulated muscle cells soon become paralyzed (nonfunctional). Affected individuals suffocate because of paralyzed respiratory muscles. 

Other types of inhibitors may not be so tolerable. Organophosphorus compounds, used in nerve gases and weed killers (e.g., parathion), form a covalent irreversible bond with the active serine and permanently inactivate acetylcholinesterase. This is a type of suicide inhibition because the inhibitor reacts with the enzyme much like a substrate, but becomes blocked in the intermediate state where the enzyme-phosphoryl bond is stable, in contrast to the hydrolyzable enzyme-acetyl bond. These compounds are life-threatening. 

As the intention was to synthesize new inhibitors of acetylcholinesterase, the anticholinesterase activity of the above-described phosphonates and phosphonothioates was studied. In 1978, Zakharova et al. examined the activity of dichlorovinyl-substituted carbaboranyl phosphonates 27-30 toward acetylcholinesterase and butyrylcholinesterase. As these compounds are analogs of 2,2-dichlorovinyl dimethyl phosphate (dichlorvos or DDVP) (26), their activity was compared with the activity of this known insecticide. As shown in Table 2.1, compounds 27-30 exhibit remarkably lower activity toward both enzymes than the phosphate DDVP. The replacement of an alkoxyl group with the bulky carborane moiety increases the steric demand of the compound and therefore hinders binding on the surface of the enzyme and the following phosphorylation of the hydroxyl group of serine. Additionally, the type of inhibition changed in snch a way that the compounds exhibit reversible instead of irreversible binding. The substituents at the second carbon atom of the carborane core have only minor influence on the inhibitor activity. 

---