Cysteine Amino Acids: Properties, Function, Benefits, and Sources

What is cysteine?

Cysteine is a naturally occurring amino acid present in limited amounts in the majority of proteins. It is the sole amino acid that possesses a thiol in its side chain. Cysteine serves as a crucial supply of sulfur in human metabolism; although categorized as a nonessential amino acid, it may be essential for babies, the elderly, and those with specific metabolic disorders. The thiol group is responsible for several critical functions of cysteine, including the formation of disulfide bonds essential for defining protein structures, stabilizing extracellular proteins, providing proteolytic resistance, and facilitating the catalytic properties of enzymes.

The amount of free cysteine within cells is carefully controlled by the liver of mammals. For example, when rats' protein and sulfur amino acid intake is altered from below-requirement to above-requirement amounts, the intracellular cysteine level remains closely within 20 and 100 nmol/g. Six hours following the introduction of the cysteine-supplemented low-protein diet, rats that had adapted to a high-protein diet showed a significant rise in portal plasma cysteine concentration, but no increase beyond the fasting value for cysteine in arterial plasma or the liver. In contrast, rats given a low-protein diet had a considerably lower concentration of cysteine in their livers but no discernible drop in plasma cysteine levels relative to fasting values. Hence, in rats, the liver permits a cysteine concentration that fluctuates by approximately five times, ranging from twenty to one hundred nanomoles per gram, while controlling the breakdown of cysteine to keep the plasma concentration of cysteine within a range of eighty to two hundred micromoles perliter. The liver satisfies two competing homeostatic demands by maintaining cysteine levels within a restricted range. Protein synthesis and the creation of other crucial chemicals, including as glutathione, coenzyme A, taurine, and inorganic sulfur, necessitate suitably high cysteine levels. However, it is equally important to maintain cysteine concentrations below the cytotoxicity threshold. Animal studies have shown that cysteine is highly toxic in excess, and human studies have linked chronically high cysteine levels to a host of diseases and conditions, including rheumatoid arthritis, Parkinson's, Alzheimer's, systemic lupus erythematosus, cardiovascular disease, and unfavorable pregnancy outcomes.

Cysteine structure

Though it shares an amino and carboxyl group with other amino acids, cysteine is distinct from all of them due to the presence of a thiol group (-SH) on its side chain. The oxidation of the thiol groups on cysteinyl residues in proteins allows them to form disulfide bonds. This helps ensure that proteins, particularly those found outside of cells, fold correctly and remain stable. One of the most important antioxidants in cell defense, glutathione, contains cysteine, which has a thiol group that reduces other molecules. In addition to its function in protein and enzyme synthesis, cysteine is a precursor of taurine and coenzyme A.

Structure of cysteine, cystine and glutathione. (Van Klaveren R J., et al., 1997)

Cysteine pKa

Cysteine is a triprotic acid with three ionizable functional groups including a carboxylic acid, an amino, and a sulfhydryl group (1.71, 8.33, and 10.78 at 25°C and close to zero ionic strength generally accepted that the first macroscopic pKa of cysteine represents the acid–base equilibria of the carboxylic acid group. Therefore, the carboxylate group at pH > 3 is fully ionized. However, the second and third macroscopic pKa values were the subject of a large debate in the 1950s through to the 1970s. Initially, Cohn and Edsall assigned the dissociation of the second proton to the ammonium group and the third to the sulfhydryl group values should represent mixed equilibrium constants. They and others (even almost 30 years later) have tried to evaluate the microscopic pintramolecular H-bonding and intermolecular H-bonding with water molecules. The microscopic pKa values) of cysteine were first determined in the classical work of Benesch and Benesch. They used UV–vis spectrophotometric titration to follow the ionization of the sulfhydryl group at 230–240 nm and assumed that the two cysteine thiolate derivatives.

Macroscopic and microscopic acid-base equilibria of cysteine. (Nagy P., et al., 2010)

Reported microscopic acid dissociation constants for cysteine. (Nagy P., et al., 2010)

Cysteine solubility

The solubility in polar protic fluids was predominantly affected by the liquids' polarity. With the exception of isopropanol and isobutanol, the solubility hierarchy of L-cysteine was water > methanol > ethanol > n-propanol > n-butanol > sec-butanol, which correlated favorably with polarity. The variability in solubility in isopropanol, isobutanol, and all polar aprotic solvents may be ascribed to numerous factors, including steric hindrance, solute–solvent interactions, solvent–solvent interactions, viscosity, and surface tension, among others.

L-cysteine exhibited limited solubility in n-butanol, isobutanol, sec-butanol, ethyl acetate, and acetonitrile at 293.15 K, with a solubility magnitude of 10^-5. Furthermore, utilizing data from all five monosolvents would significantly enhance the accuracy of the KAT-LSER model. Consequently, n-butanol, exhibiting the highest solubility among the solvents, was selected to illustrate the scenario of extremely poor solubility.

Cysteine sources

Cysteine (Cys) is an amino acid that is semi-essential for proper nutrition. The body gets l-cysteine from three different places: food absorption, the transsulfuration pathway (which starts with l-methionine degradation), and the breakdown of proteins that are already there. Because l-cysteine is quickly oxidized to l-cystine in normoxic circumstances, l-cystine is the predominant form of l-cysteine in blood, tissues, and foods. Because of the extremely lowering circumstances inside cells, l-cysteine is the most common type. Others have evaluated the literature linking oxidative stress and other clinical conditions to an imbalance of extracellular l-cysteine/l-cystine. While not all tissues have had their l-cysteine and l-cystine metabolism thoroughly investigated, prior research has shown that transportation plays a major role in maintaining a balance between the amounts of these compounds outside of cells and inside them.

(1) Strong fiber strands like hair, wool, feathers, horns, hooves, and nails are made possible by the development of disulfide bridges, which in turn contribute to the stability of proteins. Such is the case with the most typical cysteine sources. On the other hand, fruits and vegetables also contain cysteine. The different amounts of cysteine concentration found in specific fruits and vegetables by HPLC were uncovered in a study conducted by Demirkol's team. The quantities of cysteine in vegetables vary from 4 to 349 nM/g of wet weight. While red pepper had the highest concentration and avocado the lowest, carrot, potato, and broccoli did not contain any cysteine at all. There was also a significant difference between the cysteine levels in fruits and vegetables. Cysteine levels ranged from 7 nM/g wet weight in lemons to 58 nM/g wet weight in papayas. The amount of cysteine in dietary proteins is minimal, making up no more than five percent of the total amino acids. Cereal protein has 3-5 percent cysteine and legume protein has 2-4.5% cysteine, among plant proteins. In general, the cysteine content of animal proteins is higher than that of vegetable proteins.

Dietary sources of cysteine. (Vasdev S., et al., 2009)

(2) The process by which bacteria and plants produce cysteine can be broken down into three distinct phases. The first phase is assimilatory sulfate reduction, which results in reduced sulfur as sulfide. The second phase involves creating the organic backbone of cysteine, which consists of carbon and nitrogen. The third phase involves adding the reduced sulfur to the organic backbone. The conversion of inorganic sulfur into cysteine during plant cysteine biosynthesis is the most crucial step in the natural sulfur cycle. When it comes to plant cysteine biosynthesis, the two most important regulatory variables are SAT and OAS-TL. Both the availability of OAS and its role as a regulatory metabolite for the system have been proposed as important constraints on plant cysteine production. Enzymes SAT and OAS-TL have shown remarkable structural and functional conservation throughout evolution. One goal of plant cysteine production is to improve nutritional quality and other agronomic features; another is to create biopharmaceutical and bioactive compounds that plants can use for defense. From a biotechnological perspective, transgenic plants that are rich in cysteine are engineered to have better agricultural traits, such as heightened resistance to xenobiotics, heavy metals, drought, and heat. Furthermore, they may have environmental benefits in some situations, such as being more stress-tolerant, or they may be used for phytoremediation of water and soil. As far as we are aware, neither the study nor the industrial extraction of cysteine from plants has been pursued on a substantial scale.

Cysteine amino acids at Creative Peptides

Cysteine metabolism

The metabolism of cysteine is intricately associated with the metabolism of methionine. Cysteine is generated from methionine through four processes: the initial two steps are components of the methionine cycle, while the last two steps belong to the transsulfuration pathway. As previously outlined in the methionine cycle, methionine is converted into SAM, which then leads to the synthesis of homocysteine. Homocysteine is conjugated with serine in the initial step of the transsulfuration pathway by the enzyme cystathionine-β-synthase (CBS) to produce cystathionine. Cystathionine is broken by the enzyme cystathion-γ-lyase (CGL) into cysteine, 2-oxobutanoate, and ammonia in the subsequent process.

Two more processes are necessary to synthesize the GSH tripeptide from cysteine. The enzyme γ-glutamylcysteine synthetase (γGSC) catalyzes the combination of cysteine and glutamate to produce γ-glutamylcysteine. In a subsequent process facilitated by the enzyme glutathione synthetase (GSS), GSH is synthesized from γ-glutamylcysteine and glycine. GSH can undergo oxidation, therefore assisting in the preservation of redox equilibrium. In a reducing environment, such as in the presence of reactive oxygen species (ROS), a disulfide bond is established between two glutathione molecules. The linkage of the two GSH molecules results in the formation of glutathione disulfide (GSSG).

Cancer cells have elevated oxidative stress due to genetic and metabolic alterations, fast proliferation, aberrant growth, and an inflammatory microenvironment. Cancer cells require increased quantities of the antioxidant GSH, necessitating additional cysteine for its synthesis, to diminish ROS levels. If cancer cells can not acquire adequate cysteine from the extracellular environment, they experience elevated oxidative stress, which can be lethal, as they cannot meet their own antioxidant requirements. Cancer cells rely on the import of cysteine from the extracellular milieu. The import is primarily enabled by the xCT(-) antiporter, which allows cysteine to enter the cells as its dimer CSSC while glutamate is expelled into the extracellular space. The reliance of certain cancer cells on cysteine from the external environment is attributable to their reduced production of enzymes involved in the transsulfuration pathway.

Cysteine and Methionine metabolism. (Safrhansova L., et al., 2022)

Intracellular cysteine (Cys) metabolism. (Yin J., et al., 2016)

Cysteine properties

Using bioinformatics methods and databases of known or modeled protein structures, Cys, one of the least abundant amino acids, displays significantly different features when its location and distribution among homologous proteins are studied. Cys, Gly, Pro, and Trp were determined to be the four most often conserved amino acids in a study that examined the distribution of conservation levels of each residue in proteins. But a far more nuanced picture of conservation emerges. There are two extremes to the conservation pattern of Cys: regions with high levels of conservation have conservation rates of over 90%, and regions with low levels of conservation, due to high levels of degradation, have conservation rates of less than 10%. Just like Cys, only Trp (one of the other nineteen frequent amino acids) has a small population of intermediate values in terms of conservation. The high selective pressure to retain Cys or Trp residues in functionally relevant sites and to remove them from others is presumably indicated by these extreme patterns, according to Marino and Gladyshev. At functional and highly conserved locations, Trp and Cys are practically indispensable due to their hydrophobic side chains and the fact that Cys is the sole amino acid bearing thiol. Furthermore, both residues are poorly tolerated on proteins that are exposed to solvents; however, the reasons for this are different for these two amino acids, as will be explained in more detail later on.

Cys residues' physical and chemical properties are crucial to their functional features, yet it's remarkable that there's still debate on whether they should be classified as hydrophobic or not. Since Cys residues are largely buried (i.e., protected from solvents), several hydrophobicity scales classify them as hydrophobic. The physical characteristics of Cys were not the cause, according to Marino and Gladyshev; rather, it was evolutionary selection acting to reduce the frequency of unpaired surface Cys residues. The polarity of this residue is fairly similar to that of Ser, which is categorized as a polar residue, according to calculations of the partial charge distribution on each atom of Cys and similar free amino acid side chains performed using quantum mechanical techniques. Since the usual aim of mutagenesis studies is to eliminate thiol functionality while minimizing structural integrity alterations in mutant proteins, Ser and Ala are common, conservative alternatives for Cys. Considering its size, hydrogen bonding capabilities, and polarity, Ser is the most suitable candidate for this role (with a somewhat smaller oxygen substituting for the sulfur), but there are other compelling reasons to just eliminate the thiol/alcohol functional group, such in an Ala substitution. Considering both substitutions is the optimal course of action when feasible, since the structural impact of the amino acids that are replaced will differ from protein to protein.

Another characteristic that is specific to Cys residues is their propensity to be located near other Cys residues, a phenomenon called clustering in bioinformatics. This trait of Cys distribution, which can lead to disulfide bond formation, is commonly found around locations of metal binding or redox sensitivity and is found to be more prevalent in proteins expressed by animals living in harsh environments. On the other hand, structurally guided bioinformatic analyses do not regard all disulfide bonds in proteins to be "clustered" because not all of them join structurally close Cys residues when reduced. This is because of the possibility of conformational rearrangements. Cys residues that are exposed and isolated are the least conserved category for this amino acid when considering the degree of burial (away from solvent and the protein surface) and the inclination to be clustered. One possible interpretation of this discovery is that it shows an evolutionary bias against unpaired Cys residues that are visible on the surface.

The structural context in which highly polarizable cysteine residues occur in proteins varies. Because of this, their reactivity is significantly affected, which in turn affects features like accessibility and protonation state (i.e., pKa). As an illustration, α helices have a dipole moment that is more positively charged at the N-terminal end, which lowers the pKa and makes the Cys residues in these areas more reactive (see to the discussion of pKa below for additional information). In addition, the reactivity of certain proteins toward particular substrata is greatly enhanced by the presence of unique active-site designs surrounding the Cys. For instance, in peroxiredoxins, H-bonding interactions do double duty: they activate the incoming peroxide and lower the pKa of the active-site Cys. When combined with the pKa effect, which accounts for a rate of 20 M ^-1s ^-1 as seen with small-molecule thiolates, these extra features improve the overall rate enhancement, leading to reaction rates with hydrogen peroxide as high as 10⁷-10⁸ M ^-1s ^-1.

Cysteine (CYS) is the least exposed residue in proteins. (Poole L B., 2015)

Serine vs cysteine

The hydroxymethyl group (-CH2OH) exhibits polarity, rendering serine hydrophilic and soluble in water. The hydroxyl group (-OH) enables the formation of hydrogen bonding with water or other polar molecules. Serine residues are frequently phosphorylated in signaling pathways and enzyme control. The hydroxyl group (-OH) can engage in nucleophilic assaults and function as a nucleophile in enzyme catalysis.

The thiol group (-SH) is polar, albeit to a lesser extent than the hydroxyl group. The thiol group is capable of forming hydrogen bonds and exhibits strong reactivity, particularly in redox processes. Cysteine is essential for the formation of disulfide bonds through the oxidation of two thiol groups, hence stabilizing protein structures. Cysteine is also involved in antioxidant defense (as part of glutathione) and detoxification processes.

Relative binding energy and relative dissociation energy. (Pecul M., 2006)

Cysteine benefits

Thiol proteins and peptides contain the non-essential amino acid cysteine, which is involved in the production of glutathione, trypanothione, and other crucial metabolites. Coenzyme A, enzyme cofactors, and ubiquitous iron-sulfur clusters are just a few of the many crucial compounds that begin with cysteine and go on to play pivotal roles in electron transport, redox regulation, nitrogen fixation, and regulatory process sensing (). Humans can't make thiol proteins or methionine without cysteine. Many pathogens, notably trypanosomatids, rely on cysteine for cell viability, proliferation, virulence, and stress tolerance. The trans-sulfuration route allows Giardia intestinalis to produce cysteine, a critical growth factor for. The enzyme serine acetyltransferase (SAT) facilitates the conversion of acetyl-CoA to serine, resulting in -acetylserine (OAS), as part of the cysteine biosynthesis process. In the presence of sulphide donors, the next step in this reaction is catalyzed by cysteine synthase (CS), a pyridoxal 5-phosphate (PLP)-dependent protein. Parasites get cysteine from their hosts since they lack the genes necessary for cysteine manufacture.

Biomolecules containing a sulfhydryl or thiol functional group (-SH) are recognized to play an important role in essential metabolic reactions in nearly every living thing. Biothiols are essential reactive sulfur species (RSS) that are present either as standalone molecules or as components of proteins and peptides. A few examples of biothiols are homocysteine (Hcy), glutathione (GSH), and cysteine. By preventing free radical damage to cells, these biothiols provide an important antioxidant function. Specifically, disorders affecting development in children, liver function, muscular atrophy, leukemia, and atherosclerosis are associated with a deficiency in Cys. In addition, Cys is an expert in binding to ions of metals like lead, copper, mercury, and others. Cys is abundant in cancer cells in comparison to normal cells because of its critical function in cancer cell metabolism.

Cysteine functions and applications

1. Cysteine is the nearly sole metabolic entry point for reduced sulfur into cellular metabolism; it is necessary for the creation of important molecules such as biotin, coenzyme A, methionine, thiamine, or Fe/S clusters. Along with its importance in cellular activities like redox cycles, detoxification of heavy metals and xenobiotics, and metabolism of secondary metabolites, cysteine is essential for protein folding, assembly, and stability via disulfide-bond formation. Among fruits and vegetables, cysteine was discovered to possess antioxidant qualities.

2. The antidote properties of cysteine are most well-known for its capacity to mitigate the harmful effects of acetaminophen in the pharmaceutical industry. Several diseases, genetic abnormalities, and metabolic disorders, such as HIV infection and chronic obstructive pulmonary disease (COPD), have been effectively treated with it due to GSH shortage. Oral infections including gingivitis and glossitis can also be treated with cysteine.

3. The cosmetics industry uses cysteine, which may dissolve disulfide bonds in keratin, to prepare permanent hair waves instead of thioglycolic acid. An strong odor and allergic properties characterize thioglycolic, which was formerly utilized to condition hair for perms. Additionally, cystine, which is the product of two cysteine molecules, is utilized in the care of nails because it encourages healthy fingernail development, hardness, and functioning. A variety of skin care treatments, including those that combat atrophy and aging, contain acetylated cysteine (N-acetylcysteine), which is known for its safety and efficacy.

4. Adding cysteine to flour helps break down the gluten in flour, making it less sticky and easier to knead in the food industry, particularly in bakery applications. Cysteine, in theory, could enhance the dough's elasticity and cause it to rise more during baking by interfering with protein-protein interactions that occur through the formation of covalent disulphide bonds. Baking with cystein helps break down the gluten in flour, making it less sticky and easier to work with while kneading dough. Cysteine and its derivatives, like acetylcysteine, are utilized in expectorants (cough syrups) because of their strong reactivity. The bronchial mucus is liquefied because acetylcysteine breaks down the mucoproteins.

5. Animals of various kinds can get some of the sulfur-containing amino acids they need from cystine, making it a useful feed ingredient. Additionally, research suggests that L-cysteine may protect ruminant animals from nitrate poisoning.

Functions and applications of cysteine. (Ismail N I., et al., 2014)

FAQ

Is cysteine polar?

Cysteine is a polar amino acid owing to the presence of its thiol group (-SH) in the side chain, together with its amino group (-NH2) and carboxyl group (-COOH), which enhance its total polarity. The side chain and functional groups of cysteine render it hydrophilic, particularly when the thiol group is protonated (-SH). Cysteine can engage in hydrogen bond formation, hence enhancing protein solubility in aqueous settings. The polar characteristics of cysteine facilitate its interaction with other polar amino acids and solvent molecules within the protein's milieu, contributing to protein folding, stability, and enzymatic function. Moreover, the thiol group of cysteine can establish disulfide bonds (-S-S-) with other cysteine residues, which are crucial for the stabilization of protein structures, especially in extracellular proteins.

References

1. Van Klaveren R J., et al., Cellular glutathione turnover in vitro, with emphasis on type II pneumocytes, European Respiratory Journal, 1997, 10(6): 1392-1400.
2. Nagy P., et al., Redox chemistry of biological thiols[M]//Advances in molecular toxicology. Elsevier, 2010, 4: 183-222.
3. Han J., et al., Determination and Correlation of the Solubility of L-Cysteine in Several Pure and Binary Solvent Systems, Journal of Chemical & Engineering Data, 2020, 65(5): 2649-2658.
4. Safrhansova L., et al., Targeting amino acid metabolism in cancer, International review of cell and molecular biology, 2022, 373: 37-79.
5. Vasdev S., et al., The antihypertensive effect of cysteine, International Journal of Angiology, 2009, 18(01): 07-21.
6. Yin J., et al., l‐Cysteine metabolism and its nutritional implications, Molecular nutrition & food research, 2016, 60(1): 134-146.
7. Pecul M. Conformational structures and optical rotation of serine and cysteine, Chemical physics letters, 2006, 418(1-3): 1-10.
8. Poole L B. The basics of thiols and cysteines in redox biology and chemistry, Free Radical Biology and Medicine, 2015, 80: 148-157.
9. Ismail N I., et al., Production of cysteine: approaches, challenges and potential solution, International Journal of Biotechnology for Wellness Industries, 2014, 3(3): 95-101.