A7. Cysteine Chemistry - Biology

A7. Cysteine Chemistry - Biology

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Two cysteine side chains can covalently interact in a protein to produce a disulfide. Just as HOOH (hydrogen peroxide) is more oxidized than HOH (O in H2O2 has oxidation number of 1- while the O in H2O has an oxidation number of 2-) , RSSR is the oxidized form (S oxidation number 1-) and RSH is the reduced form (S oxidation number 2-) of thiols. There oxidation number are analogous since O and S are both in Group 6 of the periodic table and both are more electronegative than C.

  • A quick review of redox reactions and oxidation numbers.


When a protein folds, two Cys side chains might approach each other, and form an intrachain disulfide bond. Likewise, two Cys side chains on separate proteins might approach each other and form an interchain disulfide. Such disulfides must be cleaved, and the chains separated before analyzing the sequence of the protein. The disulfide in protein can be cleaved by reducing agents such as beta-mercaptoethanol, dithiothreitol, tris (2-carboxyethyl) phosphine (TCEP) or oxidizing agents which further oxidizes the disulfide to separate cysteic acids.

Figure: TCEP reduction of disulfides

Figure: Disulfide Oxidizing Agents - b-mercaptoethanol, dithiothreitol, and phosphines

The inside of cells are maintained in a reduced environment by the presence of many "reducing" agents, such as the tripeptide g-glu-cys-gly (glutathione). Hence intracellular proteins usually do not contain disulfides, which are abundant in extracellular proteins (such as those found in blood) or in certain organelles such as the endoplasmic reticulum and mitochondrial intermembrane space where disulfides can be introduced.

Figure: Cleaving Disulfide Bonds in Proteins

Cysteine Redox Chemistry

The sulfur in cysteine is redox-active and hence can exist in a wide variety of states, depending on the local redox environment and the presence of oxidzing and reducing agents. A potent oxidizing agent that can be made in cells is hydrogen peroxide, which can lead to more drastic and irreversible chemical modifications to the Cys side chains. If a reactive Cys is important to protein function, then the function of the protein can be modulated (sometimes reversibly, sometimes irreversibly) with various oxidizing agents, as shown in the figure below.

Figure: Redox state of Cysteine

Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

  • 1 Oncology Research Unit, Pfizer Worldwide Research and Development, San Diego, CA, USA
  • 2 Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, San Diego, CA, USA

The perception of reactive oxygen species has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g., cancer). New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically distinct alterations to the protein (e.g., sulfenic/sulfinic/sulfonic acid, disulfides). These post-translational modifications (PTM) are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

A biochemical (S_N2) reaction

One very important class of nucleophilic substitution reactions in biochemistry are the (S_N2) reactions catalyzed by (S)-adenosyl methionine (SAM) &ndash dependent methyltransferase enzymes. SAM is a coenzyme (section 6.3) that plays the role of methyl group donor: you can think of SAM in this context as being simply a methyl carbon electrophile attached to a sulfide leaving group.

There are many variations of SAM-dependent methylation reactions in nature. In the introduction to this chapter, we were introduced to a reaction occurring in bacterial DNA in which a methyl carbon is transferred from SAM to a nitrogen atom on adenine (this type of reaction is often referred to as (N)-methylation).

In the figure above, we are showing how an aspartate residue in the active site of the enzyme acts as a catalytic base: transfer of a proton from substrate to the aspartate side chain begins to enhance the nucleophilicity of the amine nitrogen as it approaches the electrophilic methyl carbon of SAM, and formation of the new (N-C) bond and cleavage of the (C-S) bond begins. These four bond-rearranging events probably take place in concerted fashion. A likely transition state is approximated below:

Of course, there are many other noncovalent interactions between active site enzyme residues and the substrate (the adenine base) and cofactor (SAM), but in the interest of clarity these are not shown. These interactions, many of which are hydrogen-bonds, help to position the adenine base and SAM in just the right relative orientation inside the active site for the nucleophilic attack to take place. (If you have access to American Chemical Society journals, a paper about an enzyme catalyzing a similar N-methylation reaction contains some detailed figures showing hydrogen-bond and charge-dipole interactions between the enzyme active site and the two substrates: see Biochemistry 2003, 42, 8394, figure 4).

The electrophile is a methyl carbon, so there is little steric hindrance to slow down the nucleophilic attack. The carbon is electrophilic (electron-poor) because it is bonded to a positively-charged sulfur, which is a powerful electron withdrawing group. The positive charge on the sulfur also makes it an excellent leaving group, because as it leaves, it becomes a neutral and very stable sulfide. All in all, we have a good nucleophile (enhanced by the catalytic base), an unhindered electrophile, and an excellent leaving group. We can confidently predict that this reaction is (S_N2). An (S_N1) mechanism is extremely unlikely: a methyl cation is very unstable and thus is not a reasonable intermediate to propose.

Notice something else about the SAM methylation mechanism illustrated in the previous figure. It is termolecular: there are three players acting in concert: the catalytic base, the nucleophile, and the electrophile. This is possible because the all three players are bound in a very specific geometry in the active site of the enzyme. In a reaction that takes place free in solution, rather than in an active site, the likelihood of three separate molecules colliding all at once, with just the right geometry for a reaction to take place, is very, very low. You should notice going forward that when we illustrate the mechanism of a reaction that takes place free in solution, we will only see bimolecular steps - two molecules colliding. Almost all of the biochemical reactions we see in this book will be enzyme-catalyzed - and termolecular steps will be common - while almost all of the laboratory reactions we see will take place free in solution, so we will only see unimolecular and bimolecular steps. (Synthetic chemists often employ non-biological catalysts that mimic enzyme active sites, but these examples are well beyond the scope of our discussion).

Think back to the acid-base chapter: the ( pK_a) of a protonated ether is approximately zero, indicating that an ether is a very weak base. Considering periodic trends in acidity and basicity, what can you say about the relative basicity of a sulfide?

Another SAM-dependent methylation reaction is catalyzed by an enzyme called catechol-(O)-methyltransferase. The substrate here is epinephrine, also known as adrenaline, and the reaction is part of the pathway by which adrenaline is degraded in the body.

Notice that in this example, the attacking nucleophile is a phenol oxygen rather than a nitrogen (that&rsquos why the enzyme is called an (O)-methyltransferase). In many cases when drawing biochemical reaction mechanisms, we use the abbreviations B: for a catalytic base and (H-A) for a catalytic acid, in order to keep the drawings from getting too 'busy' (it's also possible that the identity of the acidic or basic group may not be known).

SAM is formed by a nucleophilic substitution reaction between methionine and adenosine triphosphate (ATP). Draw a mechanism for this reaction, and explain why you chose either an (S_N1) or and (S_N2) pathway.

How Hangovers Work

A product of alcohol metabolism that is more toxic than alcohol itself, acetaldehyde is created when the alcohol in the liver is broken down by an enzyme called alcohol dehydrogenase. The acetaldehyde is then attacked by another enzyme, acetaldehyde dehydrogenase, and another substance called glutathione, which contains high quantities of cysteine (a substance that is attracted to acetaldehyde). Together, the acetaldehyde dehydrogenase and the glutathione form the nontoxic acetate (a substance similar to vinegar). This process works well, leaving the acetaldehyde only a short amount of time to do its damage if only a few drinks are consumed.

Unfortunately, the liver's stores of glutathione quickly run out when larger amounts of alcohol enter the system. This causes the acetaldehyde to build up in the body as the liver creates more glutathione, leaving the toxin in the body for long periods of time. In studies that blocked the enzyme that breaks down acetaldehyde (acetaldehyde dehydrogenase) with a drug called Antabuse, designed to fight alcoholism, acetaldehyde toxicity resulted in headaches and vomiting so bad that even alcoholics were wary of their next drink. Although body weight is a factor (see How Alcohol Works), part of the reason women should not keep up with men drink-for-drink is because women have less acetaldehyde dehydrogenase and glutathione, making their hangovers worse because it takes longer for the body to break down the alcohol.

Some of the most common hangover symptoms -- fatigue, stomach irritation and a general sense of illness all over -- can be further attributed to something called glutamine rebound. In the next section, we'll see what this aftereffect is all about.

Site-specific Fluorescent Labeling of Poly-histidine Sequences Using a Metal-chelating Cysteine

Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA2-BM. NTA2-BM is a potential fluorophore for selective tagging of proteins in vivo.

A7. Cysteine Chemistry - Biology

Amino Acids

Proteins are formed by polymerizing monomers that are known as amino acids because they contain an amine (-NH2) and a carboxylic acid (-CO2H) functional group. With the exception of the amino acid proline, which is a secondary amine, the amino acids used to synthesize proteins are primary amines with the following generic formula.

These compounds are known as a -amino acids because the -NH2 group is on the carbon atom next to the -CO2H group, the so-called carbon atom of the carboxylic acid.

The chemistry of amino acids is complicated by the fact that the -NH2 group is a base and the -CO2H group is an acid. In aqueous solution, an H + ion is therefore transferred from one end of the molecule to the other to form a zwitterion (from the German meaning mongrel ion, or hybrid ion).

Zwitterions are simultaneously electrically charged and electrically neutral. They contain positive and negative charges, but the net charge on the molecule is zero.

More than 300 amino acids are listed in the Practical Handbook of Biochemistry and Molecular Biology, but only the twenty amino acids in the table below are used to synthesize proteins. Most of these amino acids differ only in the nature of the R substituent. The standard amino acids are therefore classified on the basis of these R groups. Amino acids with nonpolar substituents are said to be hydrophobic (water-hating). Amino acids with polar R groups that form hydrogen bonds to water are classified as hydrophilic (water-loving). The remaining amino acids have substituents that carry either negative or positive charges in aqueous solution at neutral pH and are therefore strongly hydrophilic.

The 20 Standard Amino Acids

Nonpolar (Hydrophobic) R Groups
Glycine (Gly)
Alanine (Ala)
Valine (Val)
Leucine (Leu)
Isoleucine (Ile)
Proline (Pro)
Methionine (Met)
Phenylalanine (Phe)
Tryptophan (Trp)
Polar (Hydrophilic) R Groups
Negatively Charged R Groups
Aspartic acid (Asp)
Glutamic acid
Positively Charged R Groups

Use the structures of the following amino acids in the table of standard amino acids to classify these compounds as either nonpolar/hydrophobic, polar/hydrophilic, negatively charged/hydrophilic, or positively charged/hydrophilic.

Amino Acids as Stereoisomers

With the exception of glycine, the common amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisomers. The structures of the D and L isomers of alanine are shown in the figure below. Although D amino acids can be found in nature, only the L isomers are used to form proteins. The D isomers are most often found attached to the cell walls of bacteria and in antibiotics that attack bacteria. The presence of these D isomers protects the bacteria from enzymes the host organism uses to protect itself from bacterial infection by hydrolyzing the proteins in the bacterial cell wall.

A few biologically important derivatives of the standard amino acids are shown in the figure below. Anyone who has used an "anti-histamine" to alleviate the symptoms of exposure to an allergen can appreciate the role that histamine a decarboxylated derivative of histidine plays in mediating the body's response to allergic reactions. L-DOPA, which is a derivative of tyrosine, has been used to treat Parkinson's disease. This compound received notoriety a few years ago in the film Awakening, which documented it's use as a treatment for other neurological disorders. Thyroxine, which is an iodinated ether of tyrosine, is a hormone that acts on the thyroid gland to stimulate the rate of metabolism.

Acetic acid and ammonia often play an important role in the discussion of the chemistry of acids and bases. One of these compounds is a weak acid the other is a weak base.

Thus, it is not surprising that an H + ion is transferred from one end of the molecule to the other when an amino acid dissolves in water.

The zwitterion is the dominant species in aqueous solutions at physiological pH (pH 7). The zwitterion can undergo acid-base reactions, howeer, if we add either a strong acid or a strong base to the solution.

Imagine what would happen if we add a strong acid to a neutral solution of an amino acid in water. In the presence of a strong acid, the -CO2 - end of this molecule picks up an H + ion to form a molecule with a net positive charge.

In the presence of a strong base, the -NH3 + end of the molecule loses an H + ion to form a molecule with a net negative charge.

The figure below shows what happens to the pH of an acidic solution of glycine when this amino acid is titrated with a strong base, such as NaOH.

In order to understand this titration curve, let's start with the equation that describes the acid-dissociation equilibrium constant expression for an acid, HA.

Let's now rearrange the Ka expression,

take the log to the base 10 of both sides of this equation,

and then multiply both sides of the equation by -1.

By definition, the term on the left side of this equation is the pH of the solution and the first term on the right side is the pKa of the acid.

The negative sign on this right side of this equation is often viewed as "inconvenient." The derivation therefore continues by taking advantage of the following feature of logarithmic mathematics

to give the following form of this equation.

This equation is known as the Henderson-Hasselbach equation, and it can be used to calculate the pH of the solution at any point in the titration curve.

The following occurs as we go from left to right across this titration curve.

  • The pH initially increases as we add base to the solution because the base deprotonates some of the positively charged H3N + CH2CO2H ions that were present in the strongly acidic solution.
  • The pH then levels off because we form a buffer solution in which we have reasonable concentrations of both an acid, H3N + CH2CO2H, and its conjugate base, H3N + CH2CO2 - .
  • When virtually all of the H3N + CH2CO2H molecules have been deprotonated, we no longer have a buffer solution and the pH rises rapidly when more NaOH is added to the solution.
  • The pH then levels off as some of the neutral H3N + CH2CO2 - molecules lose protons to form negatively charged H2NCH2CO2 - ions. When these ions are formed, we once again get a buffer solution in which the pH remains relatively constant until essentially all of the H3N + CH2CO2H molecules have been converted into H2NCH2CO2 - ions.
  • At this point, the pH rises rapidly until it reaches the value observed for a strong base.

The pH titration curve tells us the volume of base required to titrate the positively charged H3N + CH2CO2H molecule to the H3N + CH2CO2 - zwitterion. If we only add half as much base, only half of the positive ions would be titrated to zwitterions. In other words, the concentration of the H3N + CH2CO2H and H3N + CH2CO2 - ions would be the same. Or, using the symbolism in the Henderson-Hasselbach equation:

Because the concentrations of these ions is the same, the logarithm of the ratio of their concentrations is zero.

Thus, at this particular point in the titration curve, the Henderson-Hasselbach equation gives the following equality.

We can therefore determine the pKa of an acid by measuring the pH of a solution in which the acid has been half-titrated.

Because there are two titratable groups in glycine, we get two points at which the amino acid is half-titrated. The first occurs when half of the positive H3N + CH2CO2H molecules have been converted to neutral H3N + CH2CO2 - ions. The second occurs when half of the H3N + CH2CO2 - zwitterions have been converted to negatively charged H2NCH2CO2 - ions.

The following results are obtained when this technique is applied to glycine.

Let's compare these values with the pKa's of acetic acid and the ammonium ion.

The acid/base properties of the a -amino group in an amino acid are very similar to the properties of ammonia and the ammonium ion. The a -amine, however, has a significant effect on the acidity of the carboxylic acid. The -amine increases the value of Ka for the carboxylic acid by a factor of about 100.

The inductive effect of the a -amine can only be felt at the a -CO2H group. If we look at the chemistry of glutamic acid, for example, the a -CO2H group on the R substituent has an acidity that is close to that of acetic acid.

When we titrate an amino acid from the low end of the pH scale (pH 1) to the high end (pH 13), we start with an ion that has a net positive charge and end up with an ion that has a net negative charge.

Somewhere between these extremes, we have to find a situation in which the vast majority of the amino acids are present as the zwitterion with no net electric charge. This point is called the isoelectric point (pI) of the amino acid.

For simple amino acids, in which the R group doesn't contain any titratable groups, the isoelectric point can be calculated by averaging the pKa values for the a -carboxylic acid and a -amino groups. Glycine, for example, has a pI of about 6.

At pH 6, more than 99.98% of the glycine molecules in this solution are present as the neutral H3N + CH2CO2H zwitterion.

When calculating the pI of an amino acid that has a titratable group on the R side chain, it is useful to start by writing the structure of the amino acid at physiological pH (pH 7). Lysine, for example, could be represented by the following diagram.

At physiological pH, lysine has a net positive charge. Thus, we have to increase the pH of the solution to remove positive charge in order to reach the isoelectric point. The pI for lysine is simply the average of the pKa's of the two -NH3 + groups.

At this pH, all of the carboxylic acid groups are present as -CO2 - ions and the total population of the -NH3 + groups is equal to one. Thus, the net charge on the molecule at this pH is zero.

If we apply the same technique to the pKa data for glutamic acid, given above, we get a pI of about 3.1. The three amino acids in this section therefore have very different pI values.

Thus, it isn't surprising that a common technique for separating amino acids (or the proteins they form) involves placing a mixture in the center of a gel and then applying a strong voltage across this gel. This technique, which is known as gel electrophoresis, is based on the fact that amino acids or proteins that carry a net positive charge at the pH at which the separation is done will move toward the negative electrode, whereas those with a net negative charge will move toward the positive electrode.

Chapter Five - Chimeric Small Antibody Fragments as Strategy to Deliver Therapeutic Payloads

Antibody–drug conjugates (ADCs) represent an innovative class of biopharmaceuticals, which aim at achieving a site-specific delivery of cytotoxic agents to the target cell. The use of ADCs represents a promising strategy to overcome the disadvantages of conventional pharmacotherapy of cancer or neurological diseases, based on cytotoxic or immunomodulatory agents. ADCs consist of monoclonal antibodies attached to biologically active drugs by means of cleavable chemical linkers. Advances in technologies for the coupling of antibodies to cytotoxic drugs promise to deliver greater control of drug pharmacokinetic properties and to significantly improve pharmacodelivery applications, minimizing exposure of healthy tissue.

The clinical success of brentuximab vedotin and trastuzumab emtansine has led to an extensive expansion of the clinical ADC pipeline. Although the concept of an ADC seems simple, designing a successful ADC is complex and requires careful selection of the receptor antigen, antibody, linker, and payload. In this review, we explore insights in the antibody and antigen requirements needed for optimal payload delivery and support the development of novel and improved ADCs for the treatment of cancer and neurological diseases.

<p>This section provides information on the expression of a gene at the mRNA or protein level in cells or in tissues of multicellular organisms.<p><a href='/help/expression_section' target='_top'>More. </a></p> Expression i

<p>This subsection of the 'Expression' section provides information on the expression of a gene at the mRNA or protein level in cells or in tissues of multicellular organisms. By default, the information is derived from experiments at the mRNA level, unless specified 'at protein level'.<br></br>Examples: <a href="">P92958</a>, <a href="">Q8TDN4</a>, <a href="">O14734</a><p><a href='/help/tissue_specificity' target='_top'>More. </a></p> Tissue specificity i

Manual assertion based on experiment in i

Gene expression databases

Bgee dataBase for Gene Expression Evolution

Genevisible search portal to normalized and curated expression data from Genevestigator

Organism-specific databases

Chemistry Classroom

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Watch the video: Isoelectric point and zwitterions. Chemical processes. MCAT. Khan Academy (February 2023).