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Amino Acids and Proteins

Building Bridges to Knowledge

Photo taken from the following Website: http://www.clemson.edu/centers-institutes/sullivan/patients/nutrition101/Protein/

Amino acids possess a carboxylic acid group and an amino group. The naturally occurring amino acids have the amino group on the carbon atom adjacent to the carboxylic acid group, i.e., the α carbon atom. Except for glycine, the carbon atom attached to the amine group and the carboxylic acid group is a chiral carbon atom with an “S” notation. The conventional designation of the amino acids is “α-amino acids.” Since the amine group, NH2, and the carboxylic acid group, -COOH, are in the same molecule, then the molecule could function as an inner salt. This inner salt is called a zwitterion.

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α-Amino acids have the following general structure.

The zwitterion (internal salt) structure for α-amino acids have the following general structure.

The physical properties of amino acids suggest that the zwitterion structure dominates. Amino acids are crystalline structures at room temperature, and at 200o C amino acids have a tendency to char making it difficult to determine their melting points. Amino acids have varying degrees of solubility in water; however, they are more soluble in water, a polar solvent, than nonpolar solvents such as CHCl3 and toluene. Amino acids that are found in nature have an “L” configuration. Most amino acids have an “S” notation at the chiral carbon atom where the carboxylic acid and the amine groups are attached. Following is an alphabetical listing of twenty-two (22) amino acids with their structures (the non-zwitterion format). Alanine

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IUPAC Name: (2S)-2-aminopropanoic acid Arginine

IUPAC Name: (2S)-2-amino-5-guanidinopentanoic acid Asparagine

IUPAC Name: (2S)-2-amino-3-carbamoylpropanoic acid aspartic acid

IUPAC Name: (2S)-2-aminobutanedioic acid cysteine

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IUPAC Name: (2R)-2-amino-3-sulfhydrylpropanoic acid cystine

IUPAC Name: (2R)-2-amino-3-[(2R)-2-amino-2-carboxy-ethyl]disulfanylpropanoic acid glutamine

IUPAC Name: (2S)-2-amino-4-carbamoylbutanoic acid glutamic acid

IUPAC Name: (2S)-2-aminopentanedioic acid glycine

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IUPAC Name: 2-aminoethanoic acid histidine

IUPAC Name: (2S)-2-amino-3-(1H-imidazol-4-yl)propanoic acid isoleucine

IUPAC Name: (2S, 3S)-2-amino-3-methylpentanoic acid leucine

IUPAC Name: (2S)-2-amino-4-methylpentanoic acid lysine

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IUPAC Name: (2S)-2,6-diaminohexanoic acid methionine

IUPAC Name: (2S)-2-amino-4-(methylthio)butanoic acid phenylalanine

IUPAC Name: (2S)-2-amino-3-phenylpropanoic acid proline

IUPAC Name: (2S)-pyrrolidine-2-carboxylic acid hydroxyproline

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IUPAC Name: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid serine

IUPAC Name: (2S)-2-amino-3-hydroxypropanoic acid threonine

IUPAC Name: (2S, 3R)-2-amino-3-hydroxybutanoic acid tyrosine

IUPAC Name: (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid tryptophan

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IUPAC Name: (2S)-2-amino-3-(1H-indol-3-yl)propanoic acid valine

IUPAC Name: (2S)-2-amino-3-methylbutanoic acid Glycine is the only amino acid of the twenty-two amino acids that does not rotate plane-polarized light, i.e., glycine is optically inactive. Arginine, asparagine, aspartic acid, glutamine, glutaric acid, histidine, lysine, proline, serine, threonine, tyrosine, threonine, and tyrosine have polar side chains that make them vulnerable to molecular association (hydrogen bonding). Also, amino acids with carboxylic acid side chains and amino side chains can form ion pairs. For example, if a protein contains glutamic acid and another protein contains lysine, then the two proteins could connect through a salt bridge. The following equation illustrates this concept.

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The salt bridge connects the two protein strands. Biologically important amino acids other than α-amino acids exist in nature, but they are not components of proteins. These amino acids are found in plants and some animals, including humans. For example, γ-aminobutyric acid, found in humans, is a chemical inhibitory neurotransmitter in the brain that regulates the contraction of the muscles. Also, γ-aminobutyric acid plays a role in regulating neuron excitability in many mammalian nervous systems. p-Aminobenzoic acid is used in the invivo synthesis of folic acid, one of the B complex vitamins. Human beings cannot synthesize eight of the twenty-two amino acids. These eight amino acids are phenylalanine, valine, leucine, isoleucine, methionine, threonine, tryptophan, and lysine. The eight essential amino acids must be included in the diet in order to maintain protein syntheses. Amino acids missing from the diet could cause: Kwashiorker Disease is a protein deficient disease that results in edema, ulcerating dermatoses, and an enlarged liver with fatty infiltrates.

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Marasmus Disease is a protein deficient disease that results in muscle loss and dehydration. Cachexia Disease is a protein deficient disease resulting in loss of muscle mass. Amino acids can function as acids and bases; therefore, they can act as internal buffers in living systems. The zwitterion structure of amino acids (at the isoelectric point designated by a specified pH value) can effectively scavenge small amounts of acids or small amounts of bases as illustrated by the following two equations.

The isoelectric point for an amino acid is the pH where the amino acid does not have an electric charge, and this pH is not necessarily equal to seven. The isoelectric point for an amino acid is related to the structure of the amino acid. The zwitterion structure of amino acids behave as a weak acid and a weak base where the solution may be slightly acidic, slightly basic, or neutral depending on the structure of the amino acid. Simple amino acids with no ionizable side chains have isoelectric points between pH 5.0 to pH 6.5. Basic amino acids have isoelectric points greater than 6.5, and acidic amino acids have isoelectric points less than 5.0. The net charge on the amino acid can vary depending on the pH of the solution; therefore, amino acids can migrate in an electric field at different rates. Adjusted experimental conditions, i.e., pH, can be used to separate amino acids in an electric field. This separation procedure is referred to as electrophoresis.

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Amino acids can react with nitrous acid to produce α-hydroxycarboxylic acids.

The reaction could proceed through the following mechanism. (1)

(2)

(3)

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(4)

(5)

(6)

(7)

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(8)

(9)

The addition of equations 1-9 gives

Treating amino acids with nitrous acid is a technique that can determine the number of free amino groups in a protein. This technique is referred to as the Van Slyke Method, named after its originator Donald D. Van Slyke. Ninhydrin, 2,2-dihdroxyindane-1,3-dione, is a white water soluble compound that can be used to detect the presence of amino acids. Amino acids react with ninhydrin to produce a chromophoric complex that has a purple color. The reaction, in basic media, takes place in two steps; therefore, requiring two moles of ninhydrin to react with one mole of an amino acid. The first step forms 2-hydroxyindane-1,3-dione, an aldehyde, carbon dioxide, and ammonia. The second step of the reaction involves the formation of

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the chromophore from ninhydrin, 2-hydroxyindane-1,3-dione, and ammonia. (1)

(2)

The final step is the formation of the purple colored complex by removing a proton from the chromophore formed in step 2.

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(3)

The sum of the three reactions would give an equation that explains the purple color when ninhydrin reacts with amino acids. The purple color is a positive test for the presence of amino acids.

Many students have had first hand experiences of getting purple finger and hands after working with ninhydrin. The “ε” amino group in lysine reacts with ninhydrin to form the purple complex. The color of the resulting solution varies from blue to purple. Proline and hydroxyproline give a yellow color with ninhydrin. Proteins are polymers of amino acids. Amino acids in polymers are held together by peptide bonds. Peptide bonds are amide linkages that result from the carboxylate group of one amino acid with the

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amine group of another amino acid and the splitting out of water after the peptide bond is formed.

When two amino acids form a dipeptide (as illustrated in the previous chemical equation), the dipeptide has one end with a reactive amino group attached and the other end with a reactive carboxylate group attached. A tripeptide is produced when three amino acids join to form two peptide bonds. A tetrapeptide is produced when four amino acid join to form three peptide bonds. A pentapeptide is produced when five amino acids join to form four peptide bonds. This process could continue to form multiple peptide linkages where the unreactive amino groups and the unreactive carboxylate groups react to connect many amino acids in amide linkages. Ten or more amino acids constitute a polypeptide. Protein molecules can have hundreds of amino acids with polypeptide bonds.

Polypeptides become proteins when their molecular masses exceed 10,000 g/mole. Each protein has an isoelectric point, and the protein is least soluble at its isoelectric point. Consequently, the proteins at their isoelectric point aggregate and precipitate. If the pH is at some other value other than the isoelectric point, then the protein molecules may form charge particles that may repel each other forming a colloidal dispersion.

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Human protein molecules defined as enzymes are made from twenty of the twenty-two amino acids. The missing two amino acids are cystine and hydroxyproline. Enzymes, which consist of amino acids, are biological catalyst. Enzymes lower the energy barrier to reactivity. Hydroxylation reactions introduce hydroxyl groups into molecules. Hydroxyproline is made from the hydroxylation of proline by polyl hydroxylase, an enzyme. Enzyme nomenclature includes the ending “ase.” Hydroxyproline is primarily found in protein collagen where its primary function is to stabilize collagen.

Cystine is an important amino acid used primarily in the formation of the tertiary structures of many proteins. The primary structure of proteins is the sequence of amino acids constituting the protein. Secondary structure of proteins can be described by their three-dimensional structures or the spatial arrangement of the primary structure. The spatial arrangement could be helical or pleated. The α-helical structure represented in Figure 25 A is a general example of a secondary protein structure. Tertiary structures of proteins are

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specific connections of atoms within the secondary structure where folding occurs creating a complex structure that may be spherical or rod-like. Folding may occur by salt bridges or by molecular association (hydrogen bonding). Quaternary structures are three-dimensional aggregations of two or more polypeptides each with primary structures, secondary structures, and tertiary structures. Hemoglobin is an example of the quaternary structure of proteins. Hemoglobin is an aggregate of four polypeptides where two of the polypeptides are referred to as α subunits, and the other two are referred to as β subunits. Each polypeptide is like a myoglobin molecule. Myoglobin, insoluble in water, is a fibrous protein that binds iron and oxygen, and it is found in muscle tissues of vertebrates. Myoglobin consists of 153 amino acids. Heme is a cofactor associated with the hemoglobin subunits, and it is a prosthetic group that binds oxygen and iron. Iron forms an octahedral complex with three entities- the porphyrin ring system of heme, the nitrogen of a basic amino acid in the quaternary structure of hemoglobin, and molecular oxygen.

Heme

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Serylalanycysteine (ser-ala-cys) would be an example of the primary structure for a tripeptide.

Figure 25 A represents a general example of a secondary structure of proteins (http://www.tutorvista.com/content/biology/biology-iii/cellular-macromolecules/proteins.php).

Figure 25 A Figure 25 B represents an example of tertiary and quaternary structures of proteins (http://www.tutorvista.com/content/biology/biology-iii/cellular-macromolecules/proteins.php).

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Figure 25 B The primary, secondary, tertiary, and quaternary structures of proteins are very important. Sequencing of the amino acids in their primary structures is essentially for the protein to execute its biological activity. For example, a protein with a sequence of thirty amino acids must have the thirty amino acids sequenced correctly in order to exhibit its enzymatic properties, i.e., to thermodynamically and kinetically accomplish its designated in vivo biochemical function. Incorrect sequencing of amino acids will result in errors within the biological system, e.g., inborn errors of metabolism or the inability of the biological catalyst to properly function. Some peptides and proteins can function as hormones. Hormones are various types of chemicals released in cells that affect cells of organisms that are different from the cells from which they originated. Some examples of hormones are insulin, vasopressin, and oxytocin. Insulin is a protein that plays a major role in carbohydrate metabolism. Vasopressin (compound I) and oxytocin (compound II) are peptide hormones that are found in the posterior lobe of the pituitary gland. Vasopressin, C46H65N13O12S2, (an antidiuretic hormone) increases the amount of water reabsorbed by the kidneys. It also causes an increase in blood pressure. Oxytocin, C43H66N12O12S2, controls smooth muscle contraction such as uterine contraction during childbirth.

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Vasopressin consists of nine amino acids, and oxytocin consists of nine amino acids. Vasopressin differs from oxytocin by two amino acids- phenylalanine and arginine. In oxytocin, isoleucine replaces phenylalanine and leucine replaces arginine. The proton magnetic resonance spectrum and the carbon-13 magnetic resonance spectrum of vasopressin are complicated, and by themselves, could not be the sole source of structural identification.

1HNMR Spectrum of Vasopressin

13CNMR Spectrum of Vasopressin

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1HNMR Spectrum of Oxytocin

13CNMR Spectrum of Vasopressin The magnetic resonance spectra of vasopressin and oxytocin are complicated, and would be challenging to analyze. Modern structural analyses of these complex systems can be accomplished more effectively by using sophisticated techniques such as Double Quantum Filtered Correlated Spectroscopy (DQF-COSY) and Nuclear Overhauser Effect Spectroscopy (NOESY). A brief discussion of COSY and NOESY were presented in the paper titled “Spectroscopy, Building Bridges to Knowledge.” Vincent du Vigneaud, at Cornell Medical College in New York, isolated oxytocin, identified its component amino acids, and

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synthesized the nonapeptide during the middle twentieth century. He received the 1955 Nobel Prize in Chemistry for his remarkable and extraordinary work.

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Problems Amino Acids

1. The following sequence of steps result in the formation of an amino acid. Identify the amino acid resulting from these steps.

(1)

(2)

(3)

(4)

(5)

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(6)

Suggest structures for:

(a) compound 1 (b) compound 2 (c) compound 3 (d) compound 4 (e) compound 5 (f) the amino acid

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A water soluble compound, C5H11O2 N, does not react with NaOH, but reacts with HCl to form C5H12O2 NCl. The synthesis of C5H11O2 N can be accomplished by treating glycine, the simplest amino acid, with methyl iodide, or by treating chloroacetic acid with trimethylamine. Suggest a structural formula for C5H11O2 N.

3. Suggest possible structures A, B, C,and D in the following

reactions:

(a)

(b)

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(c)

(d)

(4)

An analysis of a small heme protein gave 0.43% Fe and 1.48% S. Calculate the minimum molecular mass of this heme protein. Calculate the minimum number of iron atoms and the minimum number of sulfur atoms in a molecule of the heme protein.

(5)

A certain unknown polypeptide was shown to exhibit antibiotic properties. Steps (a) -(d) represent the complete analysis of the unknown polypeptide:

(a) The polypeptide upon hydrolysis produced the following

amino acids: leucine, ornithine, phenylalanine, proline, and valine. Ornithine is a rare amino acid, and the phenylalanine is this polypeptide has the expected “R” configuration rather than an “S” configuration at the chiral center.

D-phenylalanine

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(2R)-2-amino-3-phenylpropanoic acid

Ornithine

(b) The molecular mass of the polypeptide was determined to be approximately 1300 g/mole.

(c) Analysis for the C-terminal residue was negative. Analysis for the N-terminal residue using 2,4-dinitrofluorobenzene produced

(d) Partial hydrolysis of the polypeptide produced the following dipeptides and tripeptides:

leu-phe orn-leu phe-pro val-orn phe-pro-val pro-val-orn

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val-orn-leu

(6) Write the structure(s) of the tripeptide(s) that would be found by the sequence of m-RNA condons in (a) and (b)?

(a) UUU-UAU-ACU (b) UUU-UAC-ACU

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Solutions to Problems

1. The following sequence of steps result in the formation of an amino acid. Identify the amino acid resulting from these steps.

(1)

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(2)

32

(3)

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(4)

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(5)

(6)

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2. A water soluble compound, C5H11O2 N, does not react with NaOH, but reacts with HCl to form C5H12O2 NCl. The synthesis of C5H11O2 N can be accomplished by treating glycine, the simplest amino acid, with methyl iodide, or by treating chloroacetic acid with trimethylamine. Suggest a structural formula for C5H11O2 N.

3. Suggest possible structures A, B, C,and D in the following

reactions:

(a)

(b)

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(c)

(d)

4.

An analysis of a small heme protein gave 0.43% Fe and 1.48% S. Calculate the minimum molecular mass of this heme protein. Calculate the minimum number of iron atoms and the minimum number of sulfur atoms in a molecule of the heme protein. If one assumes that the small heme protein contains one Fe, then the minimum molecular mass would be:

If the molecule contained one sulfur atom, then the minimum molecular mass would be

0.0043 (MW) = 56.0 g/mole

MW = 56.0 g/mole0.0043

= 1.30 x 104 g/mole

0.0148 (MW) = 32 g/mole

MW = 32.0 g/mole0.0148

= 2.16 x 103 g/mole

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The number of sulfur atoms/molecule:

5.

A certain unknown polypeptide was shown to exhibit antibiotic properties. Steps (a) -(d) represent the complete analysis of this unknown polypeptide: (a) The polypeptide upon hydrolysis produced the following amino

acids: leucine, ornithine, phenylalanine, proline, and valine. Ornithine is a rare amino acid, and the phenylalanine is this polypeptide has the expected “R” configuration rather than an “S” configuration at the chiral center.

D-phenylalanine (2S)-2-amino-3-phenylpropanoic acid

Ornithine

(b) The molecular mass of the polypeptide was determined to be approximately 1300 g/mole.

(c) Analysis for the C-terminal residue was negative. Analysis for the N-terminal residue using 2,4-dinitrofluorobenzene produced

1.30 x 104

2.16 x 103 = 6

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(d) Partial hydrolysis of the polypeptide produced the following dipeptides and tripeptides:

leu-phe orn-leu phe-pro val-orn phe-pro-val pro-val-orn val-orn-leu

On the basis of the information provides in (a) - (d), suggest a structure for this polypeptide. On the basis of the information provides in (a) - (d), suggest a structure for this polypeptide. The molecular weights of the component amino acids are: leucine 131 g/mole ornithine 132 g/mole phenylalanine 165 g/mole proline 115 g/mole valine 117 g/mole

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The sum of the molecular masses is 660 g/mole The molecular mass of the peptide is approximately 1300 g/mole; therefore, one can conclude that the peptide contains two leucine amino acids, two ornithine amino acids, two phenylalanine amino acids, two proline amino acids, and two valine amino acids. The partial hydrolysis provide information of proximity amino acids, and the only amino acids exposed are the two ornithine amino acids (as determined by 2,4-dintrofluorobenzene. These data suggest the following structure for the unknown peptide:

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(6) Write the structure(s) of the tripeptide(s) that would be found by the sequence of m-RNA condons in (a) and (b)? (a) UUU-UAU-ACU (b) UUU-UAC-ACU

or

Either structure is possible, because there is no indication in the problem which amino acid is the N-terminal amino acid and which amino acid is the C-terminal amino acid.