N-Ethylmaleimide

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N-Ethylmaleimide[1]
N-ethylmaleimide.svg
Identifiers
Abbreviations NEM
CAS number 128-53-0 YesY
PubChem 4362
ChemSpider 4209 YesY
UNII O3C74ACM9V N
DrugBank DB02967
KEGG C02441 YesY
ChEBI CHEBI:44485 N
ChEMBL CHEMBL8211 YesY
Jmol-3D images Image 1
Properties
Molecular formula C6H7NO2
Molar mass 125.12528
Melting point 43-46 °C
Boiling point 210 °C (410 °F; 483 K)
Except where noted otherwise, data are given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
 N (verify) (what is: YesY/N?)
Infobox references

N-Ethylmaleimide (NEM) is an organic compound that is derived from maleic acid. It contains the imide 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]

Organic chemistry[edit]

NEM is a Michael acceptor, 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[edit]

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 as shown first by Peter Lauf in 1980 (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). This unique discovery contributed to the molecular identification of K-Cl cotransport (KCC) in human embryonic cells transfected by KCC1 isoform cDNA, some 16 years later (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). Since then NEM has been widely used as a now classic diagnostic tool to uncover or manipulate the membrane presence of K-Cl cotransport in cells of many species in the animal kingdom (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). Despite repeated unsuccessful attempts to identify chemically the target thiol group (see also: 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), it is held that, 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 (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.Cell. 2009 Aug 7;138(3):525-36. doi: 10.1016/j.cell.2009.05.031). The ensuing dephosphorylation of KCC by protein phosphatases leads to activation of KCC as first proposed by Michael Jennings in 1990 (Jennings, M. L. & Al-Rohil, N. S. J. gen. Physiol. 95, 1021−1040, 1990).

References[edit]

  1. ^ N-Ethylmaleimide at Sigma-Aldrich
  2. ^ Thiol reactive probes 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

External links[edit]

  • 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.