Thiols in proteins

GSH synthase, a tripeptide (glutamylcysteinylglycine), is not only a water-soluble antioxidant, but is also part of a redox buffer (Smith et al., 1996). It is found in all cells and is used for a multiplicity of cellular functions, such as protein and prostaglandin synthesis, detoxification, etc. Cytosolic concentrations of GSH range from 1 to 11 mm (Smith et al., 1996) and are 100-1000 times greater than the extracellular levels. Many proteins contain sulfhydryl groups because of their cysteine content. The content of thiols in proteins is greater than that of the pool of GSH (Torchinsky, 1981). 

Oxidation of thiols in proteins is often involved in regulation of enzyme activity, such as glucose-6-phosphate dehydrogenase, pyruvate kinase, brain adenylate cyclase, y-glutamyl-synthetase and others (Elstner, 1990) Carbonyl compounds can be attacked by amino groups. In-... 

In addition, nonenzymatic acylation of cysteine thiols on proteins incubated in the presence of acyl-CoA has been described, although the biological importance of this process is still unclear. 

A thiol contains an —SH group covalently bonded to carbon. Sulfur is just below oxygen in the periodic table, so a thiol is somewhat similar to an alcohol. Still, the chemical and physical properties of thiols differ significantly from those of alcohols. For example, whereas alcohols have inoffensive odors, thiols smell bad. The stench of skunk scent is due to thiols, including 3-methylbutanethiol. Thiols are important in proteins because of their abilities to form S—S linkages, which we describe in Section 13-1. 

Propiolactone is one example. It will alkylate amino, imino, hydroxyl and carboxyl groups, all of which occur in proteins, and react also with thiol and disulphide groups responsible for the secondary structure of proteins and the activity of some enzymes. 

Bolton, J. L. Turnipseed, S. B. Thompson, J. A. Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins studies utilizing amino acid and peptide models. Chem.-Biol. Interact. 1997, 107, 185-200. 

In contrast to the lability of certain dN adducts formed by the BHT metabolite above, amino acid and protein adducts formed by this metabolite were relatively stable.28,29 The thiol of cysteine reacted most rapidly in accord with its nucleophilic strength and was followed in reactivity by the a-amine common to all amino acids. This type of amine even reacted preferentially over the e-amine of lysine.28 In proteins, however, the e-amine of lysine and thiol of cysteine dominate reaction since the vast majority of a-amino groups are involved in peptide bonds. Other nucleophilic side chains such as the carboxylate of aspartate and glutamate and the imidazole of histidine may react as well, but their adducts are likely to be too labile to detect as suggested by the relative stability of QMs and the leaving group ability of the carboxylate and imidazole groups (see Section 9.2.3). 

Ou W, Silver J. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion. Virology 2006 350(2) 406-417. 

Thiolsulfonate-containing compounds can react with thiols with release of the sulfonate end of the molecule to yield disulfide derivatives. The modification reagent 2-aminoethyl-2 -aminoethanethiolsulfonate, or AEAETS, reacts with a sulfhydryl with release taurine (2-aminoethanesulfonate) to form a 2-aminoethyl-dithiol derivative (Figure 1.94). AEAETS can be used to block cysteine residues in proteins and form derivatives containing positively charged amines. 

Methyl methanethiosulfonate (MMTS) is a small reversible blocking agent for sulfhydryl groups (Thermo Fisher, Toronto Research). It reacts with free thiols to form a dithiomethane modification with release of sulfinic acid (Figure 1.122). The sulfinic acid component decomposes into volatile products, which don t affect the disulfide formed from the MMTS reaction Alkylthiosulfonates react rapidly with thiols under mild conditions at physiological pH. The MMTS compound is a liquid at 10.6 M concentration and is conveniently added to a reaction medium by pipette. Complete thiol modifications of available cysteine residues in proteins can... 

Figure 7.10 An NHS-PEG-maleimide compound can be used to functionalize dendrimers to provide a hydrophilic spacer terminating in thiol-reactive groups. Thiol-containing proteins then can be conjugated to this reactive intermediate to form covalent thioether bonds.

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