Acyl cholinesterase

Acyl Cholinesterases. Acetylcholinesterase (AChE EC 3.1.1.7 CAS 9000-81-1) is the serine esterase which catalyzes the hydrolysis of acetylcholine and possesses an esteratic site, and which is responsible for unspecific hydrolyses of several substrates. Also, butyrylcholinesterase (EC 3.1.1.8 CAS 9001-08-5) has been sometimes used for asymmetric hydrolysis of esters. Acetylcholinesterase has been used for... 

In contrast to acetylcholinesterase, cholinesterase (acylcholine acyl-hydrolase, butyrylcholinesterase, EC 3.1.1.8) exhibits relatively unspecific esterase activity toward choline esters, with abroad specificity relative to the size of the acyl group. The enzyme is synthesized in the liver and can be found in smooth muscle, adipocytes, and plasma. Its physiological role remains partly obscure, but there is evidence that it is present transiently in the embryonic nervous system, where it is replaced in later stages of development by acetylcholinesterase. It has, therefore, been suggested that cholinesterase functions as an embryonic acetylcholinesterase. 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

Most cholinesterase inhibitors inhibit the enz)nne by acylating the esteratic site on the enzyme surface. Physostigmine and neostigmine are examples of... 

The cholinesterases, acetylcholinesterase and butyrylcholinesterase, are serine hydrolase enzymes. The biological role of acetylcholinesterase (AChE, EC 3.1.1.7) is to hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline (Scheme 6.1). This plays a role in impulse termination of transmissions at cholinergic synapses within the nervous system (Fig. 6.7) [12,13]. Butyrylcholinesterase (BChE, EC 3.1.1.8), on the other hand, has yet not been ascribed a function. It tolerates a large variety of esters and is more active with butyryl and propio-nyl choline than with acetyl choline [14]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in their specificity with respect to the acyl moiety of the substrate [15]. AChE hydrolyzes ACh at a very high rate. The maximal rate for hydrolysis of ACh and its thio analog acetyl-thiocholine are around 10 M s , approaching the diffusion-controlled limit [16]. 

Succinylcholine is a neuromuscular blocking agent, which is used clinically to cause muscle relaxation. Its duration of action is short due to rapid metabolism—hydrolysis by cholinesterases (pseudocholinesterase or acylcholine acyl hydrolase)—in the plasma and liver to yield inactive products (Fig. 7.55). Thus, the pharmacological action is terminated by the metabolism. However, in some patients, the effect is excessive, with prolonged muscle relaxation and apnea lasting as long as two hours compared with the normal duration of a few minutes. 

In contrast to acetylcholinesterase, which is selective for acetylcholine, butyryl-cholinesterase tolerates a wider variety of esters and is more active with butyryl-and propionylcholines than acetylcholine [7]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in specificity to the acyl moiety of the substrate [6]. 

There is some evidence that points to an association between Alzheimer s disease and a defict in hrain acetylcholine levels. Considerable attention has thus focused on cholinesterase inhibitors as potential drugs for treating the affliction. The preparation of a relatively simple inhibitor is prepared by acylation of the benzylamine (197) with chloroformamide (198). The resulting urethane is then resolved to afford rivastigmine (199). " ... 

Cholinesterase is now considered to react with substrates and competitive inhibitors by the initial formation of an enzyme affinity complex which enables the serine hydroxyl group at the esteratic site to become acylated. The acylated group can then react with any nucleophilic reagent to regenerate the free enzyme. The overall scheme is as follows ... 

It has been shown that both the substrate acetylcholine and the inhibitor tetramethylammonium chloride serve as activators of horse plasma cholinesterase (B35), but the tetramethylammonium ion does not have any activating effect on the phosphorylation of the enzyme. Brestkin and Brick (B35) concluded that the activation process is associated with the deacylation step and not with the acylation stage. 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

Two structurally and functionally very similar, yet di.stinct enzymes form the family of cholinesterases (ChHs). Acetylcholinesterase (AChE EC 3.1.1,7) and butyryl-cholinestcrase (BuChE EC 3.1.1.8) both catalyze acetylcholine (ACh) hydrolysis with similarly high efficiency and only differ in efficiency to catalyze the hydrolysis of carboxylic acid e.sters of larger acyl group size, such as butyrylchoHnc or benzoylcholinc. Larger substrates are hydrolyzed much better by BuChE due to small but significant differences in their structure that al.so allows BuChE to... 

OPs and CMs are acylating inhibitors (ABs) of AChE and BuChE. Cholinesterases react with A6 compounds in the same way as they react with substrates that is, they acylate the hydroxyl group of serine in the catalytic site. However, there is a significant quantitative difference between substrates and AB compounds in the rates of the individual reaction steps. In the reaction with. substrates, acylation and deacylation of the serine is very fast, whereas AB compounds quickly acylate the enzyme but very slowly deacylale from the enzyme, particularly when AB is an OP. The enzyme therefore stays acylaled by AB compounds for a long time and cannot hydrolyze substrates during that time. Consequently, OP and CM compounds are inhibitors of cholinesterases. 

One group of esterases has an a,p-fold and is prominent in the liver cytosol (Quinn, 1997). Acetylcholinesterase, butyl cholinesterase, and lipases have been used as models for these esterases. Generally esterases also have amidase activity (and vice versa, due to the basic mechanisms). All esterases appear to use a catalytic triad to activate a nucleophile, which is used to form an enzyme-acyl intermediate. The triad consists of a nucleophile, a general base catalyst, and an acidic residue. 

The enzymatic nucleophile is similar in kind and reactivity to the ultimate solution acceptor. Examples of this class include the serine proteases and the alkahne phosphatase. The serine hydroxyl group is similar in chemical reactivity to the hydroxyl group of water, the final acceptor in these group transfer reactions (Fersht, 1985). For example, the active site Ser200 and His444 of cholinesterase are involved in a putative catalytic triad to effect acyl transfer (Taylor, 1991) ... 

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