Open "Back Door" in a Molecular Dynamics Simulation of Acetylcholinesterase

Gilson, Michael K.; Straatsma, Tjerk P.; McCammon, J. Andrew; Ripoll, Daniel R.; Faerman, C. H.; Axelsen, Paul H.; Silman, Israel; Sussman, Joel L.

doi:10.1126/science.8122110

Cited by 265 publications

(235 citation statements)

References 19 publications

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“…Up to 10 000 substrate molecules per second are hydrolysed in an active site that is located at the bottom of a deep and narrow gorge (Sussman et al, 1991). Based on molecular-dynamics simulations, the existence of a 'backdoor' has been postulated that could transiently open near the active site and allow rapid product clearance (Gilson et al, 1994). Several temperaturecontrolled crystallography approaches have been designed that allowed us to shed light on some aspects of the molecular traffic within AChE.…”

Section: Temperature-controlled Kinetic Cryocrystallography To Characmentioning

confidence: 99%

Temperature-dependent macromolecular X-ray crystallography

Weik

Colletier

2010

Acta Crystallogr D Biol Crystallogr

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X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

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Section: Temperature-controlled Kinetic Cryocrystallography To Characmentioning

confidence: 99%

Temperature-dependent macromolecular X-ray crystallography

Weik

Colletier

2010

Acta Crystallogr D Biol Crystallogr

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“…Alternate portals for substrate and product access have been proposed (38); however, catalytic and inhibitor-binding parameters are influenced only by mutations in the gorge (21) and not in the vicinity of the putative additional portals (39). Rapid fluctuations giving rise to transient enlargements of the gorge appear critical (40).…”

Section: Role Of the Ache Gorge Flexibility In Catalysis And Inhibitomentioning

confidence: 99%

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Bourne

Kolb

Radić

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

319

262

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The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1-and anti1-mouse AChE complexes at 2.45-to 2.65-Å resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.A cetylcholinesterase (AChE) rapidly terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter, acetylcholine, and inhibitors of AChE have been used for over a century in various therapeutic regimens (1, 2). The structure of the target enzyme reveals a narrow gorge Ϸ20 Å in depth with the catalytic triad of the active center at its base (3). Distinctive inhibitors bind to the active center or to a peripheral anionic site (PAS) located at the rim of the gorge near the enzyme surface (4-6). Previously, we generated a library of active site and PAS inhibitors with respective tacrine and phenanthridinium nuclei, each equipped with an azide or acetylene group at the end of a flexible methylene chain, to enable the reporting 1,3-dipolar cycloaddition to occur (Scheme 1) (7). AChE itself served as the reaction vessel, synthesizing its own inhibitor from these building blocks, in effect, by equilibriumcontrolled sampling of various possible pairs of reactants in its active center gorge until irreversible cycloaddition between azide and acetylene ensued at an intersecting point within the gorge, between the two anchoring positions. From 49 building block combinations, the enzyme selected the TZ2͞PA6 pair to form, with an enhanced reaction rate, a highly regioselective syn1 triazole as the sole product (Scheme 1). In contrast, chemical synthesis by t...

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“…[25] Although the situation created for observing this phenomenon (covalent tethering of catalyst and substrate) is an artificial one, this discovered mechanism of stereoselection raises the question whether nature might use similar strategies for "dual use" of active sites, providing controlled synthesis of both product enantiomers using only one enzyme. Alternative access pathways and backdoors have been suggested in key enzymes of metabolism (cytochrome P450s), [9] signal transduction (acetylcholine esterase), [26] and muscle action (myosin). [27] The regulation of different access pathways to the active center might be achieved by conformational changes or by binding of effectors, which are both standard gating mechanisms, or in the case of multienzyme complexes, alternative substrate channeling strategies might be used, thereby allowing for adaptation to changing metabolic needs.…”

mentioning

confidence: 99%

Control of Stereoselectivity in an Enzymatic Reaction by Backdoor Access

et al. 2006

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The ability of biopolymers to discriminate between optical isomers is vital for living systems, and the selective formation of only one product stereoisomer from achiral substrates is one of the most sophisticated tasks for enzymes. Using a Diels-Alder ribozyme as an example, we demonstrate here that by a simple strategy a biocatalyst can be used to selectively synthesize both product stereoisomers in one catalytic pocket, namely, by controlling access to the active site from opposite directions through different "doors". While substrates tethered to the catalyst were used to observe this phenomenon, we propose ** We gratefully acknowledge support by the Bundesministerium für Bildung und Forschung (BioFuture program), the Deutsche Forschungsgemeinschaft, and the Human Frontiers Science Program (A.J.), and by the NIH, the DeWitt Wallace Foundation, and the Abby Rockefeller Mauze Trust (D.J.P.).

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Open "Back Door" in a Molecular Dynamics Simulation of Acetylcholinesterase

Cited by 265 publications

References 19 publications

Temperature-dependent macromolecular X-ray crystallography

Temperature-dependent macromolecular X-ray crystallography

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Control of Stereoselectivity in an Enzymatic Reaction by Backdoor Access

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