Site‐directed mutants designed to test back‐door hypotheses of acetylcholinesterase function

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (⍀ loop), Cys 69 -Cys 96 , of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the ⍀ loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the ⍀ loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors. Acetylcholinesterase (AChE)1 plays a pivotal role in neurotransmission by terminating the action of neurotransmitter, acetylcholine, at neuromuscular junction and other cholinergic synapses (1-3). AChE is one of most efficient enzymes known with hydrolysis of its natural substrate reaching diffusion-controlled limits. Inhibitors of AChE target two sites in the active site gorge: an active center at the base of a narrow gorge 20 Å in depth and a peripheral site at the gorge rim (4). At the active center, a residue triad (Ser 203 -Glu 334 -His 447 ) promotes acyl transfer and hydrolysis of the substrate, whereas Trp 86 at the gorge base primarily stabilizes choline moiety of the substrate through a cationinteraction. Active site inhibitors block substrate binding either by associating with the tryptophan in the choline binding site (tacrine and edrophonium) or by reacting irreversibly with catalytic serine (carbamates and organophosphates). Peripheral site inhibitors, such as propidium and gallamine, inhibit catalytic activity through both steric blockade and allosterically altering catalytic efficiency of the active center residues (4 -8).To elucidate the conformational changes associated with mechanistically distinctive inhibitors, we developed a means for physically monitoring the conformation of purified mouse AChE by site-directed labeling with an environmentally sensitive fluorophore, acrylodan. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Cys 69 -Cys 96 omega loop (⍀ loop) flanking the active site gorge: L76C near the tip of the loop, and E81C and E...

Section: Fig 3 Fluorescence Emission Spectra Of Acrylodan-labeled Ementioning

confidence: 99%

mentioning

confidence: 99%

Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Shi

Radić

Taylor

2002

“…Modification of Glu 84 and its neighboring residues were found to have limited effect on steady-state kinetics. Faerman et al (19) inserted a cysteine at position 82 to pair with a second cysteine residing proximally in the body of the enzyme. Although it could not be firmly established that a disulfide bond formed, little change in kinetic parameters (K m and k cat ) was observed.…”

Section: Influence Of Residue Modification On Ligand Binding-mentioning

confidence: 99%

“…Crystallographic studies of the lipase revealed that the activation loop occludes the active center in the absence of substrate but folds back in the presence of lipid substrate allowing its access. Although kinetic and structural studies of AChE have not revealed evidence for such large substrate, induced lid-like movements (18,19), high catalytic turnover rates for the cholinesterases might indicate that small amplitude motions along the ⍀ loop allow rapid access of incoming substrate and release of reaction product (19). To elucidate the nature of the ligand-dependent conformational changes of AChE, we have employed cysteine substitution mutagenesis and site-directed labeling with an environmentally sensitive fluorophore, acrylodan.…”

mentioning

confidence: 99%

Reversibly Bound and Covalently Attached Ligands Induce Conformational Changes in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Shi

Boyd²,

Radić

et al. 2001

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu 76 , Glu 81 , Glu 84 , Tyr 124 , Ala 262 , and His 287 ) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (⍀ loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the ⍀ loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the ⍀ loop accompany ligand binding.Acetylcholinesterase (AChE), 1 a serine hydrolase in the ␣/␤-fold hydrolase protein superfamily (1), terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholine at a diffusion limited rate (2, 3). The crystallographic structure of mouse AChE reveals a catalytic triad (Ser 203 , Glu 334 , and His 447 ) located at the bottom of a narrow active site gorge 20 Å in depth (4 -6). Because the cross-section of the physiological substrate acetylcholine is larger than the narrowest part of the gorge, the remarkably high turnover rate of AChE raises questions regarding substrate access to the catalytic site.Molecular dynamic simulations suggest that rapid fluctuations of gorge width combined with diffusion facilitated by electrostatic forces could enhance substrate accessibility (7-10). In addition, the high affinity and slowly dissociating complex of fasciculin and AChE retains slight residual catalytic activity (11, 12), despite the occlusion of the active site gorge by fasciculin as shown in the crystal structures (5, 13, 14). Rapid fluctuations in residues lining the gorge walls may leave transient gaps at the fasciculin-AChE interface and may account for residual activity.The large omega loop (⍀ loop), Cys 69 -Cys 96 , flanking the active site gorge in mouse AChE corresponds to the activation loop ...

“…Conformational gating provides a mechanism to recruit substrates to the active site, to exclude solvent, to sequester reactive intermediates, or to enhance substrate specificity. Conformational mobility of loop Cys 69 -Cys 96 , the large ⍀ loop that is structurally homologous to the lid that sequesters the substrate in certain neutral lipases, which also show the ␣/␤-hydrolase fold (1, 2), has been proposed in gating accessibility of small molecules to the active center and in allosteric modulation of AChE catalysis (41)(42)(43). In the lipases, lid opening uncovering the catalytic site is a key feature of interfacial activation; however, the lid domain may be formed by one or more helices or surface loops that differ not only in length and sequence, but also in location relative to the ␣/␤-hydrolase core and the active site (44 -49).…”

Section: Significance Of the Structurementioning

confidence: 99%

Crystal Structure of Mouse Acetylcholinesterase

Bourne¹,

Taylor²,

Bougis³

et al. 1999

130

The crystal structure of mouse acetylcholinesterase at 2.9-Å resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short ⍀ loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking ␣-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.