Excavations into the active-site gorge of cholinesterases

Soreq, Hermona; Gnatt, Averell; Loewenstein, Yael; Neville, Lewis F.

doi:10.1016/0968-0004(92)90314-y

Cited by 59 publications

(33 citation statements)

References 17 publications

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“…This is in good agreement with the analysis of Jahr (1992), but does not provide support for the longer component of the first latency distribution predicted by the analysis of Edmonds & Colquhoun (1992). Further experiments are required to determine whether the difference between results obtained in the present experiments, and those of Edmonds & Colquhoun (1992) (Milburn et al 1991); a similar buried location for the ligand binding site has been proposed for acetylcholinesterase (Soreq, Gnatt, Loewenstein & Neville, 1992) and for nicotinic receptor ion channels (Unwin, 1993). The unusually slow association rate constant for binding of glutamate to NMDA receptors, estimated at 4-9 x 106 M-m s-' (Clements & Westbrook, 1991), would be consistent with a glutamate binding site also located in a site of restricted access.…”

Section: Discussionsupporting

confidence: 76%

Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9‐aminoacridine.

Benveniste

Mayer

1995

The Journal of Physiology

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1. N-methyl-D-aspartate (NMDA) receptor responses were recorded from rat hippocampal neurons grown in dissociated culture, using whole-cell, outside-out and nucleated patch recording techniques. Rapid perfusion was used to study voltage-dependent block of NMDA receptors by 9-aminoacridine (9-AA) and by Mg2+.2. Large amplitude tail currents were evoked on depolarization to +60 mV after application at -100 mV of NMDA and 9-AA but not NMDA and Mg2+. These tail currents were resistant to block by competitive antagonists to the glutamate and glycine binding sites on NMDA receptors and were not evoked when either NMDA or 9-AA were applied alone. 3. The decay kinetics of the tail current were dependent on agonist affinity; the time required for 80% charge transfer was 10-fold briefer for NMDA than for glutamate and 7-fold briefer for L-alanine than for glycine. These results are in accord with a sequential model for block of NMDA receptors by 9-AA, in which neither glutamate nor glycine can dissociate from the open-blocked state of the receptor. 4. Tail current responses had amplitudes 2-to 4-fold larger than responses to maximally effective concentrations of glutamate and glycine, indicating that NMDA receptor channels accumulate in the open-blocked state during co-application of agonist and 9-AA. The rise time and decay kinetics of tail current responses were faster than the response to brief applications of a maximally effective concentration of glutamate. Together, these results suggest that at +60 mV recovery from block by 9-AA occurs faster than the rate of opening of NMDA receptors in response to glutamate. 5. Our experiments suggest that open channel block of NMDA receptors can provide a novel approach for measurement of both open probability and the first latency distribution for ion channel opening in response to the binding of agonists, and provide additional evidence suggesting that the delayed opening of NMDA receptor channels underlies slow activation and deactivation of responses to glutamate.

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Section: Discussionsupporting

confidence: 76%

Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9‐aminoacridine.

Benveniste

Mayer

1995

The Journal of Physiology

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“…Lack of AcChoE modification by I is not surprising considering the three-dimensional structure of the enzyme active center (23,24). This indicates that the catalytically active serine residue is buried in a very narrow cavity.…”

Section: Resultsmentioning

confidence: 94%

Enzyme mimicry by the antiidiotypic antibody approach

Kolesnikov

Kozyr

Alexandrova

et al. 2000

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

110

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The concept of ''internal image'' of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transitionstate analog revealed structural similarity although both antibodies were elicited by two different approaches.

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“…3). (13,19) in homogenates prepared from two or three groups (two embryos per group) from a single microinjection experiment with the specified AcChoEase encoding DNA. Raw data for individual groups are expressed as A405 unit (x 10-3)/min and the net induced activity and standard deviation are presented as nmol of substrate hydrolyzed per embryo per h. Spontaneous substrate hydrolysis of 0.3 x 10-3 A405 unit/min has been subtracted.…”

Section: Resultsmentioning

confidence: 99%