N-Ethylmaleimide

Last updated
N-Ethylmaleimide [1]
N-ethylmaleimide.svg
Names
Preferred IUPAC name
1-Ethyl-1H-pyrrole-2,5-dione
Other names
Ethylmaleimide
Identifiers
3D model (JSmol)
AbbreviationsNEM
112448
ChEBI
ChEMBL
ChemSpider
DrugBank
ECHA InfoCard 100.004.449 OOjs UI icon edit-ltr-progressive.svg
EC Number
  • 204-892-4
405614
KEGG
PubChem CID
UNII
  • InChI=1S/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3 Yes check.svgY
    Key: HDFGOPSGAURCEO-UHFFFAOYSA-N Yes check.svgY
  • InChI=1/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3
    Key: HDFGOPSGAURCEO-UHFFFAOYAE
  • O=C1\C=C/C(=O)N1CC
Properties
C6H7NO2
Molar mass 125.12528
Melting point 43 to 46 °C (109 to 115 °F; 316 to 319 K)
Boiling point 210 °C (410 °F; 483 K)
Hazards
GHS labelling:
GHS-pictogram-acid.svg GHS-pictogram-skull.svg GHS-pictogram-exclam.svg
Danger
H300, H301, H311, H314, H317
P260, P261, P264, P270, P272, P280, P301+P310, P301+P330+P331, P302+P352, P303+P361+P353, P304+P340, P305+P351+P338, P310, P312, P321, P322, P330, P333+P313, P361, P363, P405, P501
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
X mark.svgN  verify  (what is  Yes check.svgYX mark.svgN ?)

N-Ethylmaleimide (NEM) is an organic compound that is derived from maleic acid. It contains the amide functional group, but more importantly it is an alkene that is reactive toward thiols and is commonly used to modify cysteine residues in proteins and peptides. [2]

Contents

Organic chemistry

NEM is a Michael acceptor in the Michael reaction, which means that it adds nucleophiles such as thiols. The resulting thioether features a strong C-S bond and the reaction is virtually irreversible. Reaction with thiols occur in the pH range 6.5–7.5, NEM may react with amines or undergo hydrolysis at a more alkaline pH. NEM has been widely used to probe the functional role of thiol groups in enzymology. NEM is an irreversible inhibitor of all cysteine peptidases, with alkylation occurring at the active site thiol group (see schematic). [3] [4]

Mechanism of irreversible inhibition of a cysteine peptidase with NEM. NEMmech.jpg
Mechanism of irreversible inhibition of a cysteine peptidase with NEM.

Case studies

NEM blocks vesicular transport. In lysis buffers, 20 to 25 mM of NEM is used to inhibit de-sumoylation of proteins for Western Blot analysis. NEM has also been used as an inhibitor of deubiquitinases.

N-Ethylmaleimide was used by Arthur Kornberg and colleagues to knock out DNA polymerase III in order to compare its activity to that of DNA polymerase I (pol III and I, respectively). Kornberg had been awarded the Nobel Prize for discovering pol I, then believed to be the mechanism of bacterial DNA replication, although in this experiment he showed that pol III was the actual replicative machinery.

NEM activates ouabain-insensitive Cl-dependent K efflux in low K sheep and goat red blood cells. [5] This discovery contributed to the molecular identification of K-Cl cotransport (KCC) in human embryonic cells transfected by KCC1 isoform cDNA, 16 years later. [6] Since then, NEM has been widely used as a diagnostic tool to uncover or manipulate the membrane presence of K-Cl cotransport in cells of many species in the animal kingdom. [7] Despite repeated unsuccessful attempts to identify chemically the target thiol group, [8] at physiological pH, NEM may form adducts with thiols within protein kinases that phosphorylate KCC at specific serine and threonine residues primarily within the C-terminal domain of the transporter. [9] The ensuing dephosphorylation of KCC by protein phosphatases leads to activation of KCC. [10]

Related Research Articles

<span class="mw-page-title-main">Protease</span> Enzyme that cleaves other proteins into smaller peptides

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling.

<span class="mw-page-title-main">DNA polymerase</span> Form of DNA replication

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction

In biology and biochemistry, protease inhibitors, or antiproteases, are molecules that inhibit the function of proteases. Many naturally occurring protease inhibitors are proteins.

<span class="mw-page-title-main">Cotransporter</span>

Cotransporters are a subcategory of membrane transport proteins (transporters) that couple the favorable movement of one molecule with its concentration gradient and unfavorable movement of another molecule against its concentration gradient. They enable coupled or cotransport and include antiporters and symporters. In general, cotransporters consist of two out of the three classes of integral membrane proteins known as transporters that move molecules and ions across biomembranes. Uniporters are also transporters but move only one type of molecule down its concentration gradient and are not classified as cotransporters.

<span class="mw-page-title-main">DNA polymerase II</span>

DNA polymerase II is a prokaryotic DNA-dependent DNA polymerase encoded by the PolB gene.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

<span class="mw-page-title-main">Cathepsin C</span> Human protease (enzyme)

Cathepsin C (CTSC) also known as dipeptidyl peptidase I (DPP-I) is a lysosomal exo-cysteine protease belonging to the peptidase C1 protein family, a subgroup of the cysteine cathepsins. In humans, it is encoded by the CTSC gene.

The Na–K–Cl cotransporter (NKCC) is a transport protein that aids in the secondary active transport of sodium, potassium, and chloride into cells. In humans there are two isoforms of this membrane transport protein, NKCC1 and NKCC2, encoded by two different genes. Two isoforms of the NKCC1/Slc12a2 gene result from keeping or skipping exon 21 in the final gene product.

<span class="mw-page-title-main">Sodium-chloride symporter</span> Protein-coding gene in the species Homo sapiens

The sodium-chloride symporter (also known as Na+-Cl cotransporter, NCC or NCCT, or as the thiazide-sensitive Na+-Cl cotransporter or TSC) is a cotransporter in the kidney which has the function of reabsorbing sodium and chloride ions from the tubular fluid into the cells of the distal convoluted tubule of the nephron. It is a member of the SLC12 cotransporter family of electroneutral cation-coupled chloride cotransporters. In humans, it is encoded by the SLC12A3 gene (solute carrier family 12 member 3) located in 16q13.

In molecular biology, the electroneutral cation-Cl family of proteins are a family of solute carrier proteins. This family includes the products of the Human genes: SLC12A1, SLC12A1, SLC12A2, SLC12A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC12A8 and SLC12A9.

<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

<span class="mw-page-title-main">Caspase 3</span> Protein-coding gene in the species Homo sapiens

Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene. CASP3 orthologs have been identified in numerous mammals for which complete genome data are available. Unique orthologs are also present in birds, lizards, lissamphibians, and teleosts.

<span class="mw-page-title-main">WNK1</span> Protein-coding gene in the species Homo sapiens

WNK , also known as WNK1, is an enzyme that is encoded by the WNK1 gene. WNK1 is serine-threonine protein kinase and part of the "with no lysine/K" kinase WNK family. The predominant role of WNK1 is the regulation of cation-Cl cotransporters (CCCs) such as the sodium chloride cotransporter (NCC), basolateral Na-K-Cl symporter (NKCC1), and potassium chloride cotransporter (KCC1) located within the kidney. CCCs mediate ion homeostasis and modulate blood pressure by transporting ions in and out of the cell. WNK1 mutations as a result have been implicated in blood pressure disorders/diseases; a prime example being familial hyperkalemic hypertension (FHHt).

<span class="mw-page-title-main">Cathepsin H</span> Protein-coding gene in the species Homo sapiens

Cathepsin H is a protein that in humans is encoded by the CTSH gene.

<span class="mw-page-title-main">Chloride potassium symporter 5</span> Protein-coding gene in the species Homo sapiens

Potassium-chloride transporter member 5 is a neuron-specific chloride potassium symporter responsible for establishing the chloride ion gradient in neurons through the maintenance of low intracellular chloride concentrations. It is a critical mediator of synaptic inhibition, cellular protection against excitotoxicity and may also act as a modulator of neuroplasticity. Potassium-chloride transporter member 5 is also known by the names: KCC2 for its ionic substrates, and SLC12A5 for its genetic origin from the SLC12A5 gene in humans.

<span class="mw-page-title-main">Chloride potassium symporter 4</span> Protein-coding gene in the species Homo sapiens

Potassium-chloride transporter, member 4 is a chloride potassium symporter protein. It is encoded by the gene SLC12A4.

<span class="mw-page-title-main">SLC12A6</span> Protein-coding gene in the species Homo sapiens

Solute carrier family 12 member 6 is a protein that in humans is encoded by the SLC12A6 gene.

<span class="mw-page-title-main">SLC12A7</span> Protein-coding gene in the species Homo sapiens

Solute carrier family 12 member 7 is a protein that in humans is encoded by the SLC12A7 gene.

DNA Polymerase V is a polymerase enzyme involved in DNA repair mechanisms in bacteria, such as Escherichia coli. It is composed of a UmuD' homodimer and a UmuC monomer, forming the UmuD'2C protein complex. It is part of the Y-family of DNA Polymerases, which are capable of performing DNA translesion synthesis (TLS). Translesion polymerases bypass DNA damage lesions during DNA replication - if a lesion is not repaired or bypassed the replication fork can stall and lead to cell death. However, Y polymerases have low sequence fidelity during replication. When the UmuC and UmuD' proteins were initially discovered in E. coli, they were thought to be agents that inhibit faithful DNA replication and caused DNA synthesis to have high mutation rates after exposure to UV-light. The polymerase function of Pol V was not discovered until the late 1990s when UmuC was successfully extracted, consequent experiments unequivocally proved UmuD'2C is a polymerase. This finding lead to the detection of many Pol V orthologs and the discovery of the Y-family of polymerases.

The cation-chloride cotransporter (CCC) family is part of the APC superfamily of secondary carriers. Members of the CCC family are found in animals, plants, fungi and bacteria. Most characterized CCC family proteins are from higher eukaryotes, but one has been partially characterized from Nicotiana tabacum, and homologous ORFs have been sequenced from Caenorhabditis elegans (worm), Saccharomyces cerevisiae (yeast) and Synechococcus sp.. The latter proteins are of unknown function. These proteins show sequence similarity to members of the APC family. CCC family proteins are usually large, and possess 12 putative transmembrane spanners (TMSs) flanked by large N-terminal and C-terminal hydrophilic domains.

References

  1. N-Ethylmaleimide at Sigma-Aldrich
  2. Thiol reactive probes Archived 2008-01-28 at the Wayback Machine at Invitrogen
  3. Nelson, D. L.; Cox, M. M. "Lehninger, Principles of Biochemistry" 3rd Ed. Worth Publishing: New York, 2000. ISBN   1-57259-153-6.
  4. Gregory, J. D. (1955) J. Am. Chem. Soc. 77, 3922-3923
  5. A chloride dependent K+ flux induced by N ethylmaleimide in genetically low K+ sheep and goat erythrocytes.P.K. Lauf and B.E. Theg. Biochem. Biophys. Res. Comm., 92:1422, 1980
  6. Gillen CM, Brill S, Payne JA, Forbush B 3rd: Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family.J Biol Chem. 1996 Jul 5;271(27):16237-44
  7. Regulation of K-Cl cotransport: from function to genes. N. C. Adragna, M. Di Fulvio and P.K. Lauf, J. Membrane Biology, 200:1-29, 2004
  8. K+ Cl Cotransport: Sulfhydryl, divalent cations and the mechanism of volume activation in a red cell. P.K. Lauf. Topical Review, J. Memb. Biol. 88:1 13, 1985
  9. Rinehart, J; Maksimova, YD; Tanis, JE; Stone, KL; Hodson, CA; Zhang, J; Risinger, M; Pan, W; Wu, D; Colangelo, CM; Forbush, B; Joiner, CH; Gulcicek, EE; Gallagher, PG; Lifton, RP (2009). "Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity". Cell. 138 (3): 525–536. doi:10.1016/j.cell.2009.05.031. PMC   2811214 . PMID   19665974.
  10. Jennings, M. L. & Al-Rohil, N. S. J. gen. Physiol. 95, 1021−1040, 1990