Sulfonium effectors of cholinesterases of different origin (comparison with ammonium analogs and action specificity)

This review for the first time combines and analyzes in detail the data on comparison of potency of sulfonium and ammonium (and other onium, in some cases) isostructural effectors of cholinesterases (substrates, irreversible organophosphorus inhibitors, and reversible inhibitors). Besides, analysis is performed of specificity of sulfonium ligands of active center towards cholinesterases of animals standing at different stages of evolutionary development.     

Flexibility versus “rigidity” of the functional architecture of AChE active center

Functional architecture of the AChE active center appears to be characterized by both structural “rigidity”, necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the “rigid” environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the “hydrophobic patch” which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the “hydrophobic patch” in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of “rigidity” and flexibility within the HuAChE gorge are an essential element of its molecular design.     

Inhibitor Analysis of Cholinesterases of Different Origin (Variation of Structure of Leaving Group of Organophosphorus Inhibitors)

There is presented an analytic review of literature data on inhibitory analysis of structure of cholinesterases of different animals (vertebrates, insects, and molluscs) performed by studying rate of their interaction with 64 organophosphorus compounds of 13 series with different structure of leaving group. The presented data are considered from the point of view of comparative biochemistry and in the light of current data on structure of the cholinesterase active center.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 3, 2005, pp. 201–216.Original Russian Text Copyright © 2005 by Moralev, Rozengart.     

Acetylcholinesterase of cobra venom. Determination of concentration of active sites]

O-Nitrophenyl dimethylcarbamate and organophosphorus inhibitors O-n-propyl-p-nitrophenyl methylphosphonate, O-n-butyl-p-nitrophenyl methylphosphonate, O,O-diethyl-p-nitrophenyl phosphate, O-n-butyl-S-(beta-ethylmercaptoethyl) methylthiophosphonate, methysulphate of O,O-diethyl-S-(beta-phenyldimethylammoniumethly) thiophosphate were used in the titration of acetylcholinesterase active site concentratration in Naja naja oxiana venom. No side reactions with the acetylcholinesterase molecule as well as with other components of the venom were observed. In titration the effective concentrations of organophosphorus inhibitors with asymmetric phosphorus were 50% of their analytical concentrations, since cobra venom cholinesterase showed practically absolute stereoselectivity against the compounds.     

Comparative Sensitivity of Cholinesterases of Different Origin to Some Irreversible Inhibitors

The analytical review is presented of literature data obtained with use of different substrates, on sensitivity of more than 90 preparations of cholinesterases from 65 different animals (vertebrate, insects, molluscs) to the most studied 6 irreversible inhibitors (eserine and 5 organophosphorus compounds). The considered data are discussed from the point of view of comparative biochemistry and in the light of current information about structure of active center of cholinesterases.     

A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds

1. It is proposed that part of a neurotoxic dose of di-isopropyl phosphorofluoridate will be covalently bound in vivo to a specific component in the brain and spinal cord as the initial biochemical event in the genesis of the lesion. 2. A test system in vitro was devised that removes many di-isopropyl phosphorofluoridate-binding sites and indicates that the specific component may be a protein present in brain at a concentration comparable with that of the cholinesterases. 3. The site was found to be present and capable of binding di-isopropyl phosphorofluoridate in vitro in brain samples taken from either normal hens or those dosed with organophosphorus esterase inhibitors that are not neurotoxic. 4. Very little of the specific binding activity was found in brain samples from hens pre-dosed with a variety of neurotoxic organophosphorus compounds. 5. A solubilized preparation of the active brain component was obtained, suitable for further purification and study.     

Insect cholinesterases and irreversible inhibitors. Statistical treatment of the data

The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.     

Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Comparative analysis of sensitivity of cholinesterases of various origin to monoonium reversible inhibitors

Analytical review of literature data has been presented about constants of interaction of cholinesterases of various animals (verterbrates and squids) with 89 onium (ammonium, phosphonium, and sulfonium) reversible inhibitors forming homologous series with regularly changed structure. Values of competitive, uncompetitive, and generalized inhibition constants have been compared. On this basis, conclusions are made about mechanism of action of the studied compounds and the predominant areas of their sorption--in the or peripheral sites of the enzymes. The presented data are discussed from the point of view of comparative biochemistry and in the light of the current information about structure of the cholinesterase active center.     

Comparative analysis of sensitivity of cholinesterases of different origin to monoonium reversible inhibitors

The analytic review of the literature data on constants of interaction of cholinesterases of different animal (vertebrates and squids) with 89 onium (ammonium, phosphonium, sulfonium) reversible inhibitors constituting homologous series with regularly varied structure is carried out. Values of the competitive, uncompetitive and generalized inhibitor constants are compared. On the basis of that, conclusions about the mechanism of action of the studied compounds and primary place of their sorption—in “anionic” or peripheral “anionic” sites of enzymes—are made. The presented data are considered from the point of view of comparative biochemistry and in light of current concepts of cholinesterase active center structure.     

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.     

Insect cholinesterases and irreversible inhibitors. Statistical treatment of data

The data on sensitivity of cholinesterases (ChE) of different insects to irreversible inhibitors, as well as the data on physicochemical parameters of amino acids constituting their active centers, were treated by factor analysis and compared. These both characteristics have been shown to be connected with the taxonomical position of the insects. There is revealed the “material substrate” of the factors responsible for the action specificity, which are specific sites in the ChE active center.     

Vanillin enones as selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII. The out of the active site pocket for the design of selective inhibitors?

New C-glycosides and α,β-unsaturated ketones incorporating the 4-hydroxy-3-methoxyphenyl (vanillin) moiety as inhibitors of carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated. The inhibition profile of these compounds is presented against four human CA (hCA) isozymes, comprising hCAs I and II (cytosolic, ubiquitous enzymes) and hCAs IX and XII (tumour associated isozymes). Docking analysis of the inhibitors within the active sites of these enzymes has been performed and is discussed, showing that the observed selectivity could be explained in terms of an alternative pocket out of the CA active site where some of these compounds may bind. Several derivatives were identified as selective inhibitors of the tumour-associated hCA IX and XII. Their discovery might be a step in the strategy for finding an effective non-sulfonamide CA inhibitor useful in therapy/diagnosis of hypoxic tumours or other pathologies in which CA isoforms are involved.     

Substrate–Inhibitor Analysis of Cholinesterase from Hemolymph of the Pacific Gastropod Mollusc Neptunea eulimata

Based on our own and literature data, a detailed substrate–inhibitor analysis of catalytic properties of cholinesterase of hemolymph of the Pacific gastropod mollusc Neptunea eulimata has been carried out. Using specific substrates and organophosphorus inhibitors, homogeneity of the enzyme preparation has been shown. The study of the substrate specificity, using 8 substrates—choline, fluorogenic and chromogenic esters—has revealed some features of similarity with mammalian erythrocyte cholinesterase. At the same time, testing of a large group (33 compounds) of organophosphorus inhibitors with various structure, including hydrophobic inhibitors studied for the first time, has established both quantitative and qualitative differences from the mammalian blood enzymes. It is concluded that cholinesterase from the neptunea hemolymph cannot be ascribed unanimously to the type of acetylhydrolases of acetylcholine (EC 3.1.1.7).     

Microbial cells as catalysts for stereoselective red–ox reactions

Enzyme catalyzed reactions are commonly used at laboratory or industrial scale. Contrarily, the whole cell catalyzed reactions are restricted to special cases. The tremendous advances in the last years in Molecular Biology and more specifically in Metabolic Engineering and Directed Enzyme Evolution have opened the door to create tailor-made microorganisms or “designer bugs” for industrial purposes. Whole cell catalysts can be much more readily and inexpensively prepared than purified enzymes and the enzymes – inside the cells – are protected from the external environment and stabilized by the intracellular medium. Three situations have traditionally been considered convenient to select the use of whole cell catalyzed processes against the free enzyme catalyzed process: i) when the enzyme is intracellular; ii) when the enzyme needs a cofactor to carry out the catalytic act and iii) in the development of multienzymatic processes. Red–ox reactions represent the molecular basis for energy generation in the cell. These reactions are catalyzed by intracellular enzymes and are cofactor dependent as red–ox reactions need electron carriers as helpers in reduction reactions (gain of electrons) or oxidation (loss of electrons).In this review we present an overview of the state of the art of red–ox biotransformations catalyzed by whole cells — wild-type or genetically engineered microorganisms. Stereoselective reductions, hydroxylations of arenes and unfunctionalized alkanes, alkene monooxygenation, and Baeyer–Villiger reactions are among the processes described along the text, focusing in their chemo-, regio- and stereoselectivity.     

The mechanism of anticholinesterase action of acetylene organophosphorus inhibitors]

Introduction of the triple bond in the leaving group of the organophosphorus inhibitor molecule gives a sharp raise of the inhibitor activity but does not change principal characteristics of the cholinesterase inhibition mechanism. The reactivation experiments suggest that inactivation of cholinesterases by these compounds occurs due to phosphorylating of the serine hydroxyl by the corresponding phosphoric acid. A close similarity was shown between acetylenic and saturated organophosphorus inhibitors in altering ka upon change of pH and tetraalkylammonium ions action. It is demonstrated that S-alkynyl esters of thioacetic acid are slowly hydrolyzed by acetylcholinesterase and cholinesterase without irreversible inhibition of the enzymes.     

New insights into the active center of rat liver cystathionase.

Treatment by urea of purified rat liver cystathionase (L-Cystathionine cysteine-lyase (deaminating), EC 4.4.1.1) provoked a similar alteration of two activities of the enzyme, namely cysteine desulfhydration and homoserine deamination. Since the decreases of the two activities were also comparable as a result of chymotrypsin digestion of the enzyme, these observations suggest that the two sites responsible for the one and the other activites are in close proximity. Studies of the effect of derivatives of substrates (S-carboxymethylcyste-ine, S-carboxyethylcysteine, S-carboxymethylhomocysteine and S-carboxyethylhomocysteine) on both activities were performed. All of them inhibited cysteine desulfhydration and homoserine deamination; in several cases, the type of inhibition was also determined. The results are in agreement with the hypothesis that each of the two sites of the active center has, at least, three binding points which "recognise" groupings of substrates or of inhibitors, and this led us to propose a model for the active center. Each site has an -NH-2 binding point, hence the active center has two -NH-2 binding points; therefore, as cystathionase consists of four subunits and contains four molecules of pyriodoxal phosphate, it might be of interest to determine whether the smallest active molecule is the dimer.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Kinetics and structure-activity relationship studies on pregnane-type steroidal alkaloids that inhibit cholinesterases

The mechanism of inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes by 23 pregnane-type alkaloids isolated from the Sarcococca saligna was investigated. Lineweaver-Burk and Dixon plots and their secondary replots showed that the majority of these compounds, that is 1, 4, 5, 6, 9, 10, 12, 13, 15-19, and 21 were found to be noncompetitive inhibitors of both enzymes. Compounds 8, 20, 22, and 23 were determined to be uncompetitive inhibitors of BChE, while compounds 11 and 14 were found to be uncompetitive and linear mixed inhibitors of AChE, respectively. Ki values were found to be in the range of 2.65-250.0 microM against AChE and 1.63-30.0 microM against BChE. The structure-activity relationship (SAR) studies suggested that the major interaction of the enzyme-inhibitor complexes are due to hydrophobic and cation-pi interactions inside the aromatic gorge of these cholinesterases. The effects of various substituents on the activity of these compounds are also discussed in details.     

Studies on the Active Site of Dihydroxy-acid Dehydratase

Several classes of substrate analogs of dihydroxy-acid dehydratase have been tested as inhibitors of this enzyme in an attempt to characterize its binding site and find what modifications in substrate structure lead to an affinity higher than that of the natural substrates. The substrate analogs were tested on dihydroxy-acid dehydratase from both spinach and Escherichia coli. One modification of the substrate that led to as much as a 1000-fold increase in binding affinity was replacement of the 3-hydroxyl group with a thiol. It has been shown previously that the 3-hydroxyl group of the substrate becomes a ligand for one Fe of the Fe-S clusters of these enzymes on binding to their active sites. It seems likely then that the tighter binding of the thiol containing analogs is due to the thiol group becoming a ligand to an iron of the Fe-S clusters of these enzymes. A second modification in substrate that led to as much as 1000-fold increase in binding affinity was the addition of a large lipophilic group. This suggests there is a large hydrophobic pocket or hydrophobic surface near the active site of dihydroxy-acid dehydratase. A modification in substrate that led to as much as a 50-fold increase in binding was the replacement of the carboxyl group of the substrate with phosphonate; however, this increase was limited to substrate analogs without a polar functionality on the carbon β to the phosphonate group. Bromopyruvate was found to irreversibly inactivate dihydroxy-acid dehydratase. Each good inhibitor we found was active on spinach dihydroxy-acid dehydratase and E. coli dihydroxy-acid dehydratase to a similar extent suggesting the active sites of the enzymes from these two organisms are similar. Some of the better inhibitors described in this report have mild herbicidal activity.