N-Ethylmaleimide
Encyclopedia
N-Ethylmaleimide 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 thiol
s and is commonly used to modify cysteine residues in proteins and peptides.
, 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).
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 bacteria
l DNA replication
, although in this experiment he showed that pol III was the actual replicative machinery.
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
that is derived from maleic acid
Maleic acid
Maleic acid is an organic compound that is a dicarboxylic acid, a molecule with two carboxyl groups. Maleic acid is the cis-isomer of butenedioic acid, whereas fumaric acid is the trans-isomer...
. It contains the imide
Imide
In organic chemistry, an imide is a functional group consisting of two carbonyl groups bound to nitrogen. These compounds are structurally related to acid anhydrides. The relationship between esters and amides and between imides and anhydrides is analogous, the amine-derived groups are less reactive...
functional group
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
, but more importantly it is an alkene
Alkene
In organic chemistry, an alkene, olefin, or olefine is an unsaturated chemical compound containing at least one carbon-to-carbon double bond...
that is reactive toward thiol
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
s and is commonly used to modify cysteine residues in proteins and peptides.
Organic chemistry
In the jargon of organic chemistryOrganic chemistry
Organic chemistry is a subdiscipline within chemistry involving the scientific study of the structure, properties, composition, reactions, and preparation of carbon-based compounds, hydrocarbons, and their derivatives...
, 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
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
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
Cysteine protease
Proteases are enzymes that degrade polypeptides. Cysteine proteases have a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic dyad. The first step is deprotonation of a thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a...
, with alkylation
Alkylation
Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene . Alkylating agents are widely used in chemistry because the alkyl group is probably the most common group encountered in...
occurring at the active site thiol
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
group (see schematic).
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 BlotWestern blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
analysis. NEM has also been used as an inhibitor of deubiquitinases.
N-Ethylmaleimide was used by Arthur Kornberg
Arthur Kornberg
Arthur Kornberg was an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid " together with Dr. Severo Ochoa of New York University...
and colleagues to knock out DNA polymerase III in order to compare its activity to that of DNA polymerase I
DNA polymerase I
DNA Polymerase I is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known...
(pol III and I, respectively). Kornberg had been awarded the Nobel Prize for discovering pol I, then believed to be the mechanism of bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
, although in this experiment he showed that pol III was the actual replicative machinery.
External links
- The MEROPSMeropsMerops may refer to:* Merops , a genus of bee-eaters.* MEROPS, an on-line database for peptidases.It may also refer to several figures from Greek mythology:* King of Ethiopia, husband of Clymene, who lay with Helios and bore Phaethon...
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.