Expression, Purification and the 1.8Å Resolution Crystal Structure of Human Neuron Specific Enolase

Chai, G.; Brewer, John M.; Lovelace, L.L.; Aoki, Takashi; Minor, Władek; Lebioda, Lukasz

doi:10.1016/j.jmb.2004.05.068

Cited by 51 publications

(68 citation statements)

References 40 publications

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“…PEP) catalyzed by the human neuronal enolase. Some preliminary enzymatic characteristics have been reported [8,15]. Activation constants for Mg 2+ and K m values for 2-PGA which we measured were in good agreement.…”

Section: Resultssupporting

confidence: 79%

“…However, the his-tagged human neuronal enolase crystallizes in a different space group and also exhibits asymmetry between subunits [8]. The data presented here use unmutated (except for the his-tag) enzymes and are not consistent with independently catalyzing subunits.…”

Section: Discussionmentioning

confidence: 72%

“…PEP) reaction are below saturation, saturating and inhibitory, respectively [2,16]. Both the human neuronal enolase and yeast enolase 1 interact with tris [8,21], so we used that buffer and also two ''non-interacting" buffers, PIPES and HEPES, for comparison. Global fits were made to data collected over the wavelength range 250-320 nm.…”

Section: Resultsmentioning

confidence: 99%

“…There is considerable crystallographic evidence [8,[22][23][24]] that binding of substrate/analogues at the active sites of both enzymes is accompanied by movement of polypeptide loops that close around the active sites. (Since crystals are routinely prepared with saturating levels of all ligands, it is not presently possible to say whether substrate binding alone -no catalytic Mg 2+ bound -produces any loop(s) movement, but it is plausible that some movement occurs [7].)…”

Section: Resultsmentioning

confidence: 99%

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Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

2010

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a b s t r a c tWe determined the kinetics of the reaction of human neuronal enolase and yeast enolase 1 with the slowly-reacting chromophoric substrate D-tartronate semialdehyde phosphate (TSP), each in tris (tris (hydroxymethyl) aminomethane) and another buffer at several Mg 2+ concentrations, 50 or 100 lM, 1 mM and 30 mM. All data were biphasic, and could be satisfactorily fit, assuming either two successive first-order reactions or two independent first-order reactions. Higher Mg 2+ concentrations reduce the relative magnitude of the slower reaction. The results are interpreted in terms of a catalytically significant interaction between the two subunits of these enzymes.

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Section: Resultssupporting

confidence: 79%

Section: Discussionmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

2010

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“…These results were later proved in Lebioda and Reed laboratories when the crystalline structures of yeast enolase and neuron-specific human enolase were established [2,45,46]. Selective mutagenesis of yeast enolase and its recent crystallographic studies revealed that the basic amino-acids Lys345, Lys396, His159, His373, and Arg374 in the catalytic center are important in the mechanism of the reversible transition of 2-PGA to PEP.…”

Section: Discussionmentioning

confidence: 88%

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Gamian

Staniszewska

Danielewicz

2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolasederived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.

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Integrated Approaches Toward High‐Affinity Artificial Protein Binders Obtained via Computationally Simulated Epitopes for Protein Recognition

Altıntaş

Takiden

Utesch

et al. 2019

Adv Funct Materials

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Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys‐Ep1) show better binding properties than those of lower stability (Cys‐Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL−1) and affinity (dissociation constant (Kd) = 5.3 × 10−11m) are achieved using Cys‐Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.

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Expression, Purification and the 1.8Å Resolution Crystal Structure of Human Neuron Specific Enolase

Cited by 51 publications

References 40 publications

Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Integrated Approaches Toward High‐Affinity Artificial Protein Binders Obtained via Computationally Simulated Epitopes for Protein Recognition

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