Acetylcholinesterase — New Roles for Gate a Relatively Long Distance to Reach the Active Site, Ache Is One of the Fastest an Old Actor Enzymes14
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PERSPECTIVES be answered regarding AChE catalysis; for OPINION example, the mechanism behind the extremely fast turnover rate of the enzyme. Despite the fact that the substrate has to navi- Acetylcholinesterase — new roles for gate a relatively long distance to reach the active site, AChE is one of the fastest an old actor enzymes14. One theory to explain this phe- nomenon has to do with the unusually strong electric field of AChE. It has been argued that Hermona Soreq and Shlomo Seidman this field assists catalysis by attracting the cationic substrate and expelling the anionic The discovery of the first neurotransmitter — understanding of AChE functions beyond the acetate product15. Site-directed mutagenesis, acetylcholine — was soon followed by the classical view and suggest the molecular basis however, has indicated that reducing the elec- discovery of its hydrolysing enzyme, for its functional heterogeneity. tric field has no effect on catalysis16.However, acetylcholinesterase. The role of the same approach has indicated an effect on acetylcholinesterase in terminating From early to recent discoveries the rate of association of fasciculin, a peptide acetylcholine-mediated neurotransmission The unique biochemical properties and phys- that can inhibit AChE17. made it the focus of intense research for iological significance of AChE make it an much of the past century. But the complexity interesting target for detailed structure–func- of acetylcholinesterase gene regulation and tion analysis. AChE-coding sequences have recent evidence for some of the long- been cloned so far from a range of evolution- a Peripheral Choline binding site suspected ‘non-classical’ actions of this arily diverse vertebrate and invertebrate binding enzyme have more recently driven a species that include insects, nematodes, fish, site profound revolution in acetylcholinesterase reptiles, birds and several mammals, among Active site research. Although our understanding of the them man. Sequence data were shortly fol- gorge additional roles of acetylcholinesterase is lowed by the first crystal model for AChE incomplete, the time is ripe to summarize the from Torpedo californica 9, which historically evidence on a remarkable diversity of had been one of the main sources of AChE for acetylcholinesterase functions. research. Later on, crystal structures from Active site O mouse10, Drosophila11 and man12 were Acetylcholine-mediated neurotransmission1,2 obtained and found to be fundamentally sim- OH –O is fundamental for nervous system function. ilar. Surprisingly for an enzyme with an extra- N NH Its abrupt blockade is lethal and its gradual ordinarily rapid catalytic reaction, the acetyl- loss, as in Alzheimer’s disease3, multiple sys- choline site was found to reside at the bottom tem atrophy4 and other conditions5, is asso- of a deep, narrow gorge (FIG. 1a). Site-directed Catalytic triad ciated with progressive deterioration of cog- mutagenesis studies13 have also delineated nitive, autonomic and neuromuscular many of the ligand-binding features of this b Ser–AChE functions. Acetylcholinesterase (AChE) enzyme, particularly a peripheral binding site HO CH O 3 AChE O hydrolyses (FIG. 1) and inactivates acetyl- that had been identified in kinetic studies and CH3 choline, thereby regulating the concentration that seems to be fundamental for some of the N+ O CH3 O– of the transmitter at the synapse (BOX 1). ‘non-classical’ functions of AChE. Acetylcholine Acetate Termination of activation is normally depen- AChE can be classified in several ways. CH3 dent on dissociation of acetylcholine from the Mechanistically, it is a serine hydrolase. Its cat- CH3 OH– N+ receptor and its subsequent diffusion and alytic site contains a catalytic triad — serine, HO CH3 O Choline hydrolysis, except in diseases where acetyl- histidine and an acidic residue (TABLE 1) — as Ser–AChE choline levels are limiting or under AChE do the catalytic sites of the serine proteases O inhibition, conditions that increase the dura- such as trypsin, several blood clotting factors, Acetyl–AChE tion of receptor activation6. and others. However, the acidic group in Figure 1 | Acetylcholinesterase. a | Structural Acetylcholine hydrolysis can also be catal- AChE is a glutamate, whereas in most other features of the enzyme. X-ray crystallography has ysed by a related, less-specific enzyme — cases it is an aspartate residue. The nucle- identified an active site at the bottom of a narrow butyrylcholinesterase (BuChE, also known ophilic nature of the carboxylate is trans- gorge, lined with hydrophobic amino-acid side chains. At the time, the catalytic triad was unique as serum cholinesterase or pseudo-cholin- ferred through the imidazole ring of histidine among serine hydrolases in having a glutamate 7 esterase) . BuChE can replace AChE by to the hydroxyl group of serine, allowing it to side chain in lieu of the familiar aspartate side hydrolysing acetylcholine and it can also act as displace the choline moiety from the sub- chain. A choline-binding site featured hydrophobic a molecular decoy for natural anti-AChEs by strate, forming an acetyl–enzyme intermedi- tryptophan residues instead of the expected reacting with these toxins before they reach ate (FIG. 1b). A subsequent hydrolysis step frees anionic groups; a peripheral binding site has also AChE8. However, AChE seems to have many the acetate group. Understanding of the cat- been identified by site-directed mutagenesis. b | The acetylcholinesterase (AChE) reaction. more functions than BuChE as, for example, alytic properties of the protein has assisted in AChE promotes acetylcholine hydrolysis by changes in levels and properties of AChE are our understanding of its inhibition by forming an acetyl-AChE intermediate with the associated with responses to numerous exter- organophosphate and carbamate inhibitors release of choline, and the subsequent hydrolysis nal stimuli. Here, we discuss our current (BOX 2). However, several questions remain to of the intermediate to release acetate. 294 | APRIL 2001 | VOLUME 2 www.nature.com/reviews/neuro © 2001 Macmillan Magazines Ltd PERSPECTIVES Crystallography and sequence analysis Box 1 | The cholinergic synapse have identified a group of related enzymes and non-catalytic proteins. Some of these are In the presynaptic neuron, choline- a transmembrane proteins with cytoplasmic acetyltransferase (ChAT) catalyses the Presynaptic neuron domains and extracellular AChE-homologous synthesis of acetylcholine (ACh) from choline ChAT Acetyl-CoA domains that share the unique topography of and acetyl-coenzyme A (panel a). ACh is vAChT ACh + choline AChE and its strong electric field (TABLE 1)18. packaged in synaptic vesicles via a vesicular On the basis of their structures, all of these ACh transporter (vAChT). Action potentials High-affinity (–) choline uptake are classified as α/β-fold proteins; on the trigger the release of ACh into the synaptic K+ basis of their electric fields, they are classified cleft, where ACh can bind to muscarinic as electrotactins19. receptors located on the pre- and postsynaptic AChE genes from different species are membrane. Muscarinic M2 receptors (M2) on the presynaptic membrane regulate ACh Choline organized and sequentially spliced in a man- + acetate release via a negative feedback response. At the ner associated with distinct domains in their postsynaptic site, M1 receptors transduce ACh protein products. They include sites for alter- signals through a pathway involving AChE-R native splicing of the pre-mRNA both at the 22 diacylglycerol (DAG), inositol-1,4,5- 5′ (REFS 20,21) and 3′ ends . Alternative splic- 2+ trisphosphate (Ins(1,4,5)P3) and a Ca - ing allows the production of three distinct dependent protein kinase (PKC). In the + 2+ AChE variants, each with a different carboxy- hippocampus, most of the postsynaptic K Ins(1,4,5)P3 Ca terminal sequence — the ‘synaptic’ (S),‘ery- receptors are of the M1 subtype; in the cortex cAMP DAG PKC throcytic’ (E) and ‘readthrough’ (R) AChE M2 receptors might also be located on the Postsynaptic site isoforms. The carboxy-terminal sequences postsynaptic membrane. Genomic disruption determine their homologous assembly into of the M1 receptor impairs the activation of ACh M1 M2 AChE-S vAChT AChE oligomers and their heterologous asso- several signal-transduction pathways and ciation with non-catalytic subunits that explains why muscarinic excitation is the direct the subcellular localization of the pro- primary cause of seizures106. ACh is hydrolysed b Cortex Hippocampus tein. In AChE-S, a cysteine located three in the synaptic cleft by AChE-S tetramers, residues from the carboxyl terminus of the which are indirectly attached to the human protein allows dimerization by disul- neuromuscular junction by a collagen-like phide bridging. Two additional monomers tail5, or by another structural subunit to brain 107 can become associated by hydrophobic inter- synapses . AChE-R monomers would remain actions23. These tetramers can attach cova- soluble within the synaptic cleft. A high- lently to a hydrophobic P subunit or to a col- affinity choline-uptake mechanism returns lagen-like protein known as the T subunit24. choline to the presynaptic neuron. Brain distribution of AChE includes both The collagen-like subunit has a polyproline acetylcholine-releasing and cholinoceptive sequence that can form a triple-helical struc- neurons. Panel b shows a cranial section of a Striatum Amygdala ture that bundles together 4, 8 or 12 AChE-S brain stained for AChE