Indian Journal of Biochemistry & Biophysics Vo!. 39, April 2002, pp. 77-81

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Unusual catalytic activities and functions of cholinesterases

A S Balasubramanian

Department of Biotechnology, Bharathiar University, Coimbatore 641046, Tamilnadu, India

Received 1 February 2002

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) have been shown to exhibit not only esterase activity but also an amine sensitive aryl acylamidase and a metallo-carboxypeptidase activities. There is also evidence to indicate that they have functions in the substantia nigra of brain, in neural cell differentiation, cell division and tumorigenesis, cell­adhesion and detoxication mechanisms. Butyrylcholinesterase is suggested to act as a back-up enzyme in acetylcholinesterase knock-out mice. Cholinesterases have catalytic or non-catalytic roles in these functions. Partial sequence homology to many other proteins having different functions and a metal binding site which can influence functions are probably factors that confer the non-cholinergic functions and activities on cholinesterases.

Cholinesterases are the enzymes that catalyze the cleavage by hydrolysis of choline esters. The role of acetylcholinesterase (AChE), the most important among the group in the hydrolysis of acetyl choline, the neurotransmitter required for cholinergic neurotransmission, is well established. Butyryl­cholinesterase (BChE), which preferentially acts on butyryl choline was recognized as the most abundant cholinesterase in human serum whose function continues to be under investigation.

Techniques of molecular cloning, sequence ana­lysis and X-ray crystallography have helped elucida­tion of the amino acid sequence of both AChE and BChE and the three dimensional structure of AChEI.2. The a-/~-hydrolase fold found in the cholinesterases and several other enzymes has given rise to the concept of a cholinesterase family of e.nzymes3

.

Sequence conservation analysis relates the cholinesterases to a superfamily of polypeptides, including enzymes like carboxylesterases, cholestero­lesterase and other esterases, lipases, thyroglobulin and cell adhesion proteins4

. Partial sequences of different proteins occurring in the polypeptide chain of one single protein increase the possibility of a protein exhibiting more than one biological activity. Interesting observations of a protein exhibiting two different catalytic activities depending upon the type of metal ion bound to it or due to structural alterations have been reported5

•6

. It is against this background that the new activities and functions attributed to AChE and BChE, described below, have to be viewed.

E-mail : balab@vsn!.net

Amine sensitive aryl acylamidase activity exhibited by AChE and BChE

Apart from the esterase activity, both AChE and BChE exhibit aryl acylamidase activity7-9. This aryl acylamidase (AAA) activity consists in the cleavage of o-nitroacetanilide to o-nitroaniline and acetate. The interesting aspect of AAA activity of both AChE and BChE is that it is inhibited specifically by 5-hydroxy tryptamine (5-HT). This amine sensitivity of AAA has been demonstrated using AChE and BChE from different sources4

.7

-9

. On the other hand, the AAA activity of BChE alone is activated several fold by tyramine8

.9

. Interestingly, the tyramine activation of AAA is seen only with BChE from human and monkey serum but not with serum BChE from other animals lO

• It is noteworthy that immunological identity to human BChE is shown only by monkey BChEIO

•11

.

Several lines of evidence have been presented to show that the AAA activity and cholinesterase activity are localized in the same polypeptide fragment of BChE I2

. Moreover, both cholinesterase and AAA activities are inhibited in an identical fashion by typical cholinesterase inhibitors and both activities are co-immunoprecipitated by antibodies raised against AChE or BChE7,8,13.

In search of a possible physiological role for AAA The most likely function of the 5-HT sensitive

AAA is the deacetylation or deacylation of compounds with physiological or pharmacological importance, Acetyl or acyl groups have an important role in analgesia l4

. Heroin obtained by acetylating both the hydroxyl groups of morphine is more

78 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 39, APRIL 2002

euphoria-producing than morphine.The N-acetyl group in analgesic drugs like phenacetin or paracetamol reduces their toxicity and enhances their usefulness 14. Aspirin is known to acetylate the hydroxyl group of an internal serine residue in the enzyme prostaglandin cyclooxygenase causing its inactivation and thus produce its analgesic action 15.

The endogenous analgesic peptide endorphin is known to occur as N-acetylated endorphin (acetylated on its N-terminal tyrosine) in an inactive form in porcine pitui tary l6. Anandamide (N-arachidonyl ethanolamine) in the brain binds to and activates cannabinoid receptors l7

. An endogenous amidohydro­lase activity is implicated in the catalysis of the cleavage of anandamide to ethanolamine and arachidonate.

A role for the AAA in any of the above mentioned deacylation mechanisms can be envisaged. Of particular relevance to the 5-HT sensitivity of AAA is the known fact that serotonergic neurons are involved in analgesia and low concentrations of 5-HT in neurons is associated with high responsiveness to pain and to stimuli, in general 18

. It is also noteworthy that an excess of tyramine, a stimulator of AAA associated with human serum BChE, is correlated with migraine l9

. Thus, a role for AAA in the control of amine sensitive pain mechanisms through its action on endogenous acylamido compounds modulated by 5-HT and tyramine remains a possibility.

Indeed, BChE has been demonstrated to hydrolyse . . d . d . . h 2021 It Id aspmn an cocame an mactIvate t em '. wou

be of interest to find out the catalytic sites in BChE involved in these reactions and whether biological amines have any effect on these reactions.

The widespread use of synthetic pesticides which inhibit cholinesterases and the natural occurrence of cholinesterase inhibitors in some plants ( eg, eserine in the calabar bean, solanine in green potato peel and cocaine in cocoa plant) demands a mechanism by which humans who consume plant products can detoxify these compounds. It is possible that the occurrence of cholinesterases in some human tissues and body fluids where their function is not apparent is intended for detoxication and protection from cholinesterase inhibitors22

.

A metallo-carboxypeptidase activity in BChE Esterase and amidase activities are exhibited by

several proteases such as trypsin, chymotrypsin, carboxypeptidase A, elastase and thrombin. Cholinesterases have been shown to exhibit protease activity by different investigators from time to time.

Some of the earlier claims of proteolytic aCtIVIty found in cholinesterases were later shown to be due to

.. 4 contammatmg enzymes .

Human serum BChE has been shown to act as a metatallo-carboxypeptidase. The inseparability of the peptidase from the purified BChE was observed under all the conditions tried including immunoprecipitation using poly- or mono- clonal antibodies I2

.13

. The peptidase activity was stimulated by zinc and other metal ions and inhibited by metal chelators23

.

Moreover, a zinc binding site involving His-X-X­Glu .... His (found in carboxypeptidases and several other zinc binding enzymes) was identified in BChE4,23,

The crystal structure of AChE (which has a 53% sequence identity to BChE) shows a striking similarity to the overall structure of wheat serine carboxypeptidase le, Further, a great deal of similarity in the folds of wheat serine carboxy­peptidase 11 and the zinc exopeptidase, carboxypepti­dase A has also been reported24

,

Limited chymotryptic digestion of purified BChE followed by Sephadex chromatography showed two fragments, one exhibiting cholinesterase and AAA activities and the other showing peptidase activity, indicating a separate location of the catalytic site for peptidase activity, In accordance with this was the observation that, classical inhibitors of BChE which affect the active site serine residue did not inhibit carboxypeptidase activity l2,

Immunoprecipitation of BChE free of proteolytic activity has been reported. Presumably, pH of 3.9 used in the experiment might have resulted in the loss of protease activity as it was reported that the cholinesterase activity itself lost nearly 60% of its activity under this condition25

.

Co-existence of metallo-carboxypeptidase and aminoacylase activities in other enzymes

Since BChE which shows co-existence of aryl acylamidase or N-deacetylase actIVIty and a carboxypeptidase activity, it is of interest to know the presence of other such enzymes. A bifunctional zinc­dependant metallo-enzyme structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase has been described. Some of its characteristics suggest that carboxypepti­dase and aminoacylase might have evolved from a common origin26

. Another zinc stimulated metallo­enzyme exhibits N-acetyl tyrosine deacetylase and N­acetyl enkephalin carboxypeptidase activities27

. A

BALASUBRAMANIAN: UNUSUAL CATALYTIC ACTIVITIES AND FUNCTIONS OF CHOLINESTERASES 79

model given for pancreatic carboxypeptidase A shows that it can also act as an N -deacety lase28

.

Is there any physiological function for the carboxypeptidase activity in cholinesterases?

The physiological significance of the metallo­carboxypeptidase activity in BChE remains elusive. Also, no detailed investigations have been undertaken to find out whether AChE has a metallo­carboxypeptidase activity, although a zinc binding site in AChE has been reported29

. Abnormal cholinesterases, similar to BChE, have been found associated wi th the amyloid plaques in Alzheimer's disease3o. Since the major defect in Alzheimer's disease is an abnormal proteolytic mechanism resulting in amyloid ~-peptide deposition in the plaques, one may speculate that the peptidase activity of BChE or an abnormal cholinesterase may have a link to this disease. It is known that cholinesterase inhibitors are the drugs of choice in the treatment of Alzheimer's disease.

Cholinesterases have been implicated in neuronal cell differentiation and cell proliferation (see sections below). It is likely that the peptidase activity plays a role in these processes.

Cholinesterases as cell adhesion molecules A structural or sequence similarity between

cholinesterases and certain cell adhesion molecules have given rise to speculation that cholinesterases may be involved in adhesion properties. Two Drosophila adhesion proteins, glutactin and neurotactin have structural similarities to cholinesterases31

,32 . Two more proteins, neuroligin and gliotactin have also been demonstrated to have

. '1' h l' 3334 sequence sImI anty to c 0 mesterases ' . The homology between cholinesterases and

adhesion molecules also extends to their glycosylation. For example, an epitope, HNK-l carbohydrate epitope, a common motif of many cell adhesion molecules is found in certain AChE species35

. Moreover a non-catalytic form of serum BChE has also been shown to contain an O-linked HNK-l carbohydrate epitope36

. Since HNK-l epitopes have been linked to adhesion functions, cell differentiation and migration processes, the possibility of their involvement in the postulated role of cholinesterases in neuronal development and differentiation exists37

.

It is interesting to note that AChE accelerates fibrillation of amyloid ~-peptide and promotes

amyloid deposition in plaques in the Alzheimer brain . A peripheral anionic site of AChE was shown involved in this process . BChE which lacks the peripheral site did not affect amyloid formation 38

. On the other hand, BChE has been shown bound to amyloid ~-peptide fibrils in Alzheimer's disease, especially at the later stages of plaque formation suggesting that the participation of BChE sets the stage for the onset of clinical dementia39

.

Cholinesterases in neurogenetic processes and cell proliferation

Histochemical work by Layer and group has shown that both AChE and BChE are expressed in crucial times and places during early neurogenesis suggesting that they could be involved in regulating these processes40

,4I .

High BChE activities are typical of cells in transient state shortly before final mitosis and the beginning of the cellular differentiation (cells from the neural crest, adult glia and BChE producing cells of the eye margin). From studies using cholinesterase inhibitors it was concluded that a secondary or peripheral site of cholinesterases was involved in neurite growth. Intense levels of AChE activity was noticed in some developing thalamocortical projections during early postnatal development42

. It was speculated that a protease activity associated with AChE aids the ingrowth of thalamocortical neurons to penetrate through the cortical neurophil to reach their target cells43

.

It is known that cholinesterase genes are structurally altered and their protein products are aberrantly expressed in a variety of tumour types. Soreq and her group envisaged a molecular mechanism that may link cholinesterases to the cell division process44

,45. Consistent with the idea that BChE may have a role in cell proliferation, it was observed that in several SV40 transformed cell lines, BChE activity was increased many fold, whereas the parental untransformed cells had no detectable BChE

. . RNA46 actIVIty or m .

AChE in substantia nigra Behavioural and electrophysiological effects of

AChE (that does not involve its catalytic activity) has been observed in the substantia nigra of the brain. An ATP-sensitive potassium channel abundant in substantia nigra and important in nigral cellular and system functioning appeared to be involved in the action of AChE47

.

80 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, APRIL 2002

Table I-Proposed non-cholinergic activities and functions of chol i nesterases

Cholinesterases Biological activity

AChE, BChE Amine sensitive aryl acylamidase activity Neural cell differentiation Cell division and tumorigenesis Cell adhesion and cell-cell interaction

AChE Functions in substantia ni gra

BChE Metallocarboxypeptidasc-like activity Detox ication mechanisms Back-up cnzyme for AChE

A function for BChE in the AChE knock-out mouse In a surprising observation, it was found that the

AChE knock-out mouse developed to term in utero despite the absence of AChE activity in all the tissues and survived for several weeks48

. When BChE activity was examined in the tissues of the knock-out mice as well as normal mice it was found to be abundant in most of the tissues. The use of Triton x­lOO for tissue extraction was found inhibitory to BChE and this was suggested as the reason for the low tissue levels of BChE reported by earlier workers. These findings of an abundance of BChE in tissues combined with the observation that AChE is not essential for life suggest that BChE serves as a back­up to AChE in supporting and regulating cholinergic transmission 48.

Conclusions From what appeared as simple ester hydrolyzing

enzymes, the cholinesterases have come a long way to identify themselves as enzymes capable of multiple functions (Table1). The existence of a large number of variants of plasma BChE (at least twenty genotypes are known at present and perhaps more remain to be identified) and the different types of substrates (butyryl choline and other choline esters, 0-

nitroacetanilide, cocaine, aspirin and peptides such as enkephalin and Phe-Leu) acted upon by it have added a new dimension to the research interest in BChE. Cholinesterases seem to have functions both in their catalytic and non-catalytic roles. The true significance of some of these catalytic activities and functions should await further studies.

Acknowledgement A research grant from the Council of Scientific and

Industrial Research (CSIR), New Delhi is gratefully acknow ledged.

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