Dov Barak

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the ‘active site gorge’ of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the ‘gorge’, and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase

The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analogm-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000–11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000–6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500–460-fold) differed from that toward the phosphates (12–95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7–5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket.

The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors

The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.

Electrostatic Attraction by Surface Charge does not Contribute to the Catalytic Efficiency of Acetylcholinesterase

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP‐conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30 100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP‐conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Homology Model-Assisted Elucidation of Binding Sites in GPCRs

G protein-coupled receptors (GPCRs) are important mediators of cell signaling and a major family of drug targets. Despite recent breakthroughs, experimental elucidation of GPCR structures remains a formidable challenge. Homology modeling of 3D structures of GPCRs provides a practical tool for elucidating the structural determinants governing the interactions of these important receptors with their ligands. The working model of the binding site can then be used for virtual screening of additional ligands that may fit this site, for determining and comparing specificity profiles of related receptors, and for structure-based design of agonists and antagonists. The current review presents the protocol and enumerates the steps for modeling and validating the residues involved in ligand binding. The main stages include (a) modeling the receptor structure using an automated fragment-based approach, (b) predicting potential binding pockets, (c) docking known binders, (d) analyzing predicted interactions and comparing with positions that have been shown to bind ligands in other receptors, (e) validating the structural model by mutagenesis.

Selective Signaling via Unique Ml Muscarinic Agonistsa

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AP102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the ml AChR subtype. Notably, the selectivity of the new ml agonists is reflected also by activation of select second messenger systems via distinct G‐proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand‐selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimers disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Structural Modifications of the Ω Loop in Human Acetylcholinesterase

Conformational mobility of the surface Ω loop (Cys‐69‐Cys‐96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen‐bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the Ω loop in the HuAChE is not involved in large lid‐like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp‐86 supporting the proposed coupling between the structure of the Ω loop and the positioning of the Trp‐86 indole moiety, in catalytic activity and in allosterism.

Accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions

The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimers disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.

Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray-ionization mass spectrometry

While non‐reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post‐inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl‐phosphonofluoridates, were measured by electrospray‐ionization mass spectrometry (ESI‐MS). The measured mass of the free enzyme was 64 700 Da (calculated 64 695 Da) and those of the methylphosphono‐HuAChE adducts, bearing isopropyl, isobutyl, 1,2‐dimethylpropyl and 1,2,2‐trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64 780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2‐dimethylpropyl methylphosphono‐HuAChE, the change in the molecular mass and the kinetics of non‐reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to ‘aging’ of phosphonyl‐AChE adducts.