N-Ethylmaleimide

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]

N-Ethylmaleimide[1]
Names
Preferred IUPAC name
1-Ethyl-1H-pyrrole-2,5-dione
Other names
Ethylmaleimide
Identifiers
3D model (JSmol)
Abbreviations NEM
112448
ChEBI
ChEMBL
ChemSpider
DrugBank
ECHA InfoCard 100.004.449
EC Number
  • 204-892-4
405614
KEGG
UNII
  • InChI=1S/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3 checkY
    Key: HDFGOPSGAURCEO-UHFFFAOYSA-N checkY
  • 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:
GHS05: CorrosiveGHS06: ToxicGHS07: Exclamation mark
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).
☒N verify (what is checkY☒N ?)
Infobox references

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.

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]

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
  • The MEROPS online database for peptidases and their inhibitors: NEM
  • The bifunctional analogues such as p-NN'-phenylenebismaleimide can be used as cross-linking reagent for cystine residues. see Lutter, L. C., Zeichhardt, H., Kurland, C. G. & Stoffier,G. (1972) Mol. Gen. Genet. 119, 357-366.
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