Iodoacetate cysteine residues

For deterrnination of tryptophan, 4 M methanesulfonic acid hydrolysis is employed (18). For cystine, the protein is reduced with 2-mercaptoethanol, the resultant cysteine residue is carboxymethylated with iodoacetic acid, and then the protein sample is hydroly2ed. Also, a one-pot method with mercaptoethanesulfonic acid has been developed for tryptophan and cystine (19). 

Problem 26.10 1 Show the structure of the product you would expect to obtain by SN2 reaction of a cysteine residue with iodoacetic acid. 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Carboxymethylation. It was found by Vallee and Li that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate.1405 Since this reaction causes deactivation of the enzyme, this cysteine residue, later identified as Cys-46,1406 was suggested to be at the active site. The deactivated carboxymethylated enzyme still binds NAD+. The carboxymethylation of this residue is preceded by a reversible binding of iodoacetate to the enzyme.1407 This observation has helped to identify an anion-binding site in the coenzymebinding domain, where the pyrophosphate group of the coenzyme binds. 

Thus, iodoacetate has the highest reactivity toward sulfhydryl cysteine residues and may be directed specifically for—SH modification. If iodoacetate is present in limiting... 

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

Specific modification of Cys-46. Li and Vallee 86,87) and Harris 86) found that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate. The modified enzyme is inactivated and this cysteine residue, Cys-46 92), was suggested to be at the active site of the enzyme. The same residue in the S subunit is also especially reactive 20,94). The modification is preceded by anion binding of the iodoacetate and stimulated by the presence of imidazole 140,142,142). By using these facts and working with the crystalline enzyme, it is possible to achieve a highly specific and complete modification (ISO). X-ray studies of the carboxymethylated enzyme and the reaction mechanism of this modification are described in Section II,H. The carboxymethylation has been used to establish that both the EE 19) and SS 20) isozymes are active in u oxidations of fatty acids. 

Two of the cysteine residues are especially reactive toward chemical modification. Thus, one residue per subunit is selectively alkylated with iodoacetate (55) and a different one with butylisocyanate 406,407). In both cases the enzyme is inactivated and protected by the coenzyme against modification, suggesting that these residues are at the active sites of the enzyme. The two residues are now known 12,137) to be homologous to the two reactive cysteine residues in the horse enzyme, Cys-46 and Cys-174 (Section II,E,l,a), which are ligands to the active site zinc atom (Section II,C,3,b). A number of other reagents, apart from reactive coenzyme analogs, have also been shown to modify essential cysteine residues, i.e., probably either of these residues. Thus, one cysteine residue... 

Piszkiewicz and Smith (295) attempted to determine whether any of the cysteine residues were specifically reactive with iodoacetate. With the C-labeled reagent, the reaction was stopped at various times of alkylation. At various degrees of reaction, tryptic peptide patterns were examined by two-dimensional electrophoresis and chromatography. At each... 

Write the mechanism for the reaction of a cysteine residue with iodoacetic acid. 

A special alkylation reagent is iodoacetic acid. This compound reacts with mercapto groups as for example in cysteine residues ... 

When wool proteins are dissolved by alkaline solutions [13,242] of reducing agents, they contain cysteine residues. To prevent reoxidation of the cysteine residues to cystine residue disulfide cross-links by dissolved oxygen, the dissolved reduced wool proteins are usually treated with iodoacetate ions, which react with the cysteine residues. 

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