Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second order rate constants for substrate hydrolysis (kE), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme-substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for kE that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k2, a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T.

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“…1 at low initial substrate concentration [S] 0 ( i.e. , [S] 0 ≤ ~0.2 K app ) (22), as given in eq. 2.…”

Section: Experimental Methodsmentioning

confidence: 99%

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Auletta

Johnson

Rosenberry

2010

Chemico-Biological Interactions

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“…In addition, fasciculins appear not to totally occlude access of small molecules to the catalytic site (Marchot et al, 1993;Radii et al, 1995). Rather, they influence AChE catalysis in an allosteric fashion, although a partial gating influence may also restrict the rate of entry into the gorge for substrates whose catalysis is rate-limited or near-limited by diffusion (Eastman et al, 1995;RadiC et al, 1995;van den Born et al, 1995).…”

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confidence: 99%

Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin

Marchot

Ravelli²,

Raves

et al. 1996

Protein Science

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A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculinmAChE complex. Complexes of two distinct fasciculins, Fasl-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6,22 or P6,22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a V,, value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of FasZAChE complex (72 kDa) per asymmetric unit. The complex Fasl-mAChE crystallizes in the same space group with identical cell dimensions.

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“…Our results differ from these groups only in relation to Al 3+ and Li + . The main peripheral anionic site in AChE is described as a region near the rim of the gorge where the active center is located [21]. Nevertheless, there are binding sites for positively charged activators and inactivators far from the active site of the enzyme which are different for organic and inorganic molecules [13].…”

Section: Discussionmentioning

confidence: 99%

Effect of ions on the activity of brain acetylcholinesterase from tropical fish

Assis¹,

Linhares²,

Oliveira³

et al. 2015

J Coast Life Med

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Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation

Cited by 11 publications

References 15 publications

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin

Effect of ions on the activity of brain acetylcholinesterase from tropical fish

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