Cholinesterases substrate hydrolysis

Natural (-)-cocaine (7.57, Fig. 7.8), which has the (2/ ,3S)-configuration, is a relatively poor substrate for hepatic carboxylesterases and plasma cholinesterase (EC 3.1.1.8), and also a potent competitive inhibitor of the latter enzyme [116][121], In contrast, the unnatural enantiomer, (+)-(2S,3/ )-cocaine, is a good substrate for carboxylesterases and cholinesterase. Because hydrolysis is a route of detoxification for cocaine and its stereoisomers, such metabolic differences have a major import on their monooxygenase-catalyzed toxification, a reaction of particular effectiveness for (-)-cocaine. 

Smith, J. C., and Foldes, F. F., The recognition of atypical plasma cholinesterase by relative substrate hydrolysis rate. Biochim. Biophys. Acta 289, 352-358 (1972). 

The hydrolysis of cholinesterase substrates proceeds in three steps ... 

A number of papers describe tedmiques for determination of cholinesterase activity based on amperometric measurement of products formed as a result of enzymatic hydrolysis (equation 1). In this case, artificial (butyryl or acetyl thiocholine) cholinesterase substrates are used. Thiocholine, formed as a result of cholinesterase-catalyzed hydrolysis can be measured amperometrically on a platinum electrode (14, 15) or mercury electrode (16). Analyses based on thiocholine determination employing an electrode modified by cobalt phthalocyanine (17-22) or cobalt tetraphenylporphyrin (23) have been described. Enzymatic hydrolysis of... 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

Observations The preliminary treatment of the cholinesterase-containing material with allelochemical (or other compound, e.g. active oxygen species, ozone free radicals and peroxides, formed in allelopathic relations) is for 30 min, then a substrate acetylcholinesterase is added to the reaction medium and final reaction of hydrolysis is for 1 h. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

Whereas the above evidence clearly points to a catalytic activity of serum albumin, it does not exclude an activity toward less-reactive substrates due to contamination of some HSA preparations. Indeed, the hypothesis of a contamination by plasma cholinesterase (EC 3.1.1.8) has been raised [126][127]. The efficient hydrolysis of nicotinate esters by HSA (see Chapt. 8) [128][129] could be due to contamination by cholinesterase in samples of a commercially available, essentially fatty acid free albumin. Support for this hypothesis was obtained when HSA contaminated with cholinesterase was resolved into two peaks by affinity chromatography, and the esterase activity toward nicotinate esters was found exclusively in the cholinesterase fraction [130],... 

Enol esters are distinct from other esters not because of a particular stability or lability toward hydrolases, but due to their hydrolysis releasing a ghost alcohol (an enol), which may immediately tautomerize to the corresponding aldehyde or ketone. A well-studied example is that of vinyl acetate (CH3-C0-0-CH=CH2), a xenobiotic of great industrial importance that, upon hydrolysis, liberates acetic acid (CH3-CO-OH) and acetaldehyde (CH3-CHO), the stable tautomer of vinyl alcohol [25], The results of two studies are compiled in Table 7.1, and demonstrate that vinyl acetate is a very good substrate of carboxylesterase (EC 3.1.1.1) but not of acetylcholinesterase (EC 3.1.1.7) or cholinesterase (EC 3.1.1.8). The presence of carboxylesterase in rat plasma but not in human plasma explains the difference between these two preparations, although the different experimental conditions in the two studies make further interpretation difficult. 

Several drugs, in particular neuropharmacological agents, feature a car-boxylate group esterified to an aminoalkyl moiety. As a rule, such lipophilic compounds are good substrates for hydrolases, and their duration of action is influenced by their rate of hydrolysis (see also Sect. 7.3.4). A simple example is that of procaine (7.56), which is rapidly inactivated by hydrolysis [41] [76a], Various hydrolases catalyze the reaction, in particular plasma cholinesterase and cellular carboxylesterases. As often reported, atropine and scopolamine are rapidly hydrolyzed by plasma carboxylesterases in rabbits (with very large differences between individual animals), but are stable in human plasma [1] [75] [76a] [110]. 

Simple alkyl or aryl thioesters are commonly assayed as substrates of hydrolases, witness the hydrolysis of phenyl thioesters by horse serum carbox-ylesterase [150], For most substrates investigated, e.g., phenyl thioacetate, phenyl thiopropionate, and phenyl thiobutyrate (7.66, R = Me, Et, and Bu, respectively), kcat values were found, which were a few times larger than those of corresponding nitrophenyl esters, whereas the affinities were lower by approximately one order of magnitude. Methyl and phenyl esters of various linear thioacids were also found to be good substrates of mammalian liver carboxylesterases and serum cholinesterases [151]. 

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]. 

This enzyme [EC 3.1.1.8] (also known as cholinesterase, pseudocholinesterase, acylcholine acylhydrolase, nonspecific cholinesterase, and benzoylcholinesterase) catalyzes the hydrolysis of an acylcholine to generate choline and a carboxylic acid anion. A variety of choline esters and a few other compounds can serve as substrates. 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

The extensive studies on substrate and inhibitor specificity, on kinetics of hydrolysis, and on the influence of pH variations on the reactions catalyzed by cholinesterases have given very instructive information on the structure of the active surface and the mechanism of enzymatic hydrolysis. The conclusions reached in the various chapters of the present dis-... 

A very sensitive and commonly used method for cholinesterase determination was described by Ellman et al. (1961), based on hydrolysis of the thiochohne substrates acetyl- and butyrylthiocholine or others. After enzymatic hydrolysis, the relevant acid and thiochohne are released... 

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... 

Acetylcholinesterase (AChE) (also termed true cholinesterase ) is found in the synaptic cleft of cholinergic synapses, and is of undoubted importance in regulation of neurotransmission by rapid hydrolysis of released endogenous acetylcholine (ACh). AChE is also found in erythrocytes and in the CSF, and can be present in soluble form in cholinergic nerve terminals, but its function at these sites is not clear, AChE is specific for substrates that include acetylcholine and the agents methacholine and acetylthiocholine. but it has little activity with other esters. It has a maximum turnover rate at very low concentrations of AChE (and is inhibited by high concentrations). 

The colorimetric method is based on the hydrolysis of the substrate acetylthiocholine to acetate and thiocholine as performed by the cholinesterase. Thiocholine is then reacted with 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow anion (5-thio-2-nitrobenzoate). The latter is quantitated by spectrometric analysis at 405 nm, with the concentration being proportional to the cholinesterase activity in the given sample. Also for a few days postmortem the cholinesterase activity in different tissues is measurable. ... 

Beckett and his co-workers (B14) examined the hydrolysis by purified horse serum cholinesterase of a number of analogs of butyrylcholine. The introduction of an a-methyl group into the choline moiety of the butyryl ester was found to decrease the rate of enzymic hydrolysis only slightly, whereas a drastic reduction was found for d-methyl substitution. The substitution of various functional groups into the butyryl moiety of butyrylcholine modified the affinity of the substrate for the enzyme. These results are summarized in Table 3. Since values can be assumed to be inversely proportional to affinity values, the results shown in this table indicate that j8-hydroxyl substitution increases the affinity of the substrate for the enzyme, and a,/3-unsaturation effects even greater affinity. Acetoacetylcholine has a greater affinity for the enzyme surface... 

The foregoing studies have dealt chiefly with model substrates in vitro. Several of the early papers by Augustinsson, referred to in Section 4.1.1, considered substrate specificity from the viewpoint of species variations. It is also important to recognize that plasma cholinesterase may be associated with the hydrolysis, in vivo, of a large number of drugs (K4, LI, L4) that contain ester bonds susceptible to enzymic hydrolysis. Apart from succinylcholine (Section 3.1), cholinesterase is known to be responsible in man for the hydrolysis of cocaine (S40), procaine (K2), and other esters with local anesthetic properties. Whether enzymatic hydrolysis terminates the pharmacologic effect depends on the whole mechanism of action of the particular drug. 

Horse and human plasma cholinesterases have been found to have similar substrate specificity profiles (M25), for example, in respect to the relative rates of hydrolysis of some isomeric substrates (B14), as summarized in Table 4. It can be assumed that the areas in the regions of the... 

Hase (H16) studied the effect of pH on the hydrolysis of acetylcholine by horse serum cholinesterase, and his results have been reanalyzed by Laidler (L5) and extensively discussed by Dixon and Webb (D21). The relationship between pH and the rate of hydrolysis of acetylcholine has been used to obtain information on the structure of the active site of the enzyme (B19, W28). Acetylcholine is a particularly suitable substrate for these studies since it does not change its charge in the pH range studied. Similar pH-activity curves have been obtained using other substrates for cholinesterase (H23, S20, P19). Moreover the pH dependence of enzymic activity varies with the buffer system (K3). By investigating the effect of pH and sodium chloride concentration on the rate of hydrolysis of ben-zoylcholine by human plasma cholinesterase, Kalow (K6) deduced that for this substrate, each enzyme molecule contains at least two binding sites which differ in their dependence on pH. Michaelis constants and maximum hydrolysis velocities were measured for each of the two binding sites, and pK values of the enzyme-substrate complexes were found to be 5.2, 6.7, and 9.2 for one site, and 5.2, 7.0, 8.4, and 8.8 for the other. 

The basis of our current understanding of the two types of active site which are present in cholinesterase has been provided by Wilson and Bergmann (W30), who substantiated the concept of anionic and esteratic sites introduced by Zeller and Bisegger (Z2). Although it is now generally accepted that the hydrolysis of the ester bond of the substrate occurs at the esteratic site, there is still some uncertainty regarding the existence of the anionic site in butyrylcholinesterase an alternative site has been proposed by Augustinsson (A28). 

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