The three kinds of polymers that are prevalent in nature are

23 Amino Acids, Peptides, and Proteins The three kinds of polymers that are prevalent in nature are polysaccharides, proteins, and nucleic acids. You have already learned about polysaccharides, which are naturally occurring polymers of sugar subunits (ection 22.18), and nucleic acids are covered in hapter 27. We will now look at proteins and the structurally similar, but shorter, peptides. Peptides and proteins are polymers of amino acids linked together by amide bonds. The repeating units are called amino acid residues. Amino acid polymers can be composed of any number of monomers. A dipeptide contains two amino acid residues, a tripeptide contains three, an oligopeptide contains three to 10, and a polypeptide contains many amino acid residues. Proteins are naturally occurring polypeptides that are made up of 40 to 4000 amino acid residues. From the structure of an amino acid, we can see that the name is not very precise. The compounds commonly called amino acids are more precisely called a-amino- carboxylic acids. amide bonds 3 -aminocarboxylic acid amino acids are linked together by amide bonds an amino acid oxidized glutathione Proteins and peptides serve many functions in biological systems. ome protect organisms from their environment or impart strength to certain biological structures. air, horns, hooves, feathers, fur, and the tough outer layer of skin are all composed largely of a structural protein called keratin. ollagen, another structural protein, is a major component of bones, muscles, and tendons. ome proteins have other protective functions. nake venoms and plant toxins, for example, protect their owners from other species, blood-clotting proteins protect the vascular system when it is injured, 959

960 APTE 23 Amino Acids, Peptides, and Proteins and antibodies and protein antibiotics protect us from disease. A group of proteins called enzymes catalyzes the chemical reactions that occur in living systems, and some of the hormones that regulate these reactions are peptides. Proteins are also responsible for many physiological functions, such as the transport and storage of oxygen in the body and the contraction of muscles. 23.1 lassification and omenclature of Amino Acids 3-D Molecules: ommon naturally occurring amino acids The structures of the 20 most common naturally occurring amino acids and the frequency with which each occurs in proteins are shown in Table 23.1. ther amino acids occur in nature, but only infrequently. All amino acids except proline contain a primary amino group. Proline contains a secondary amino group incorporated into a five-membered ring. The amino acids differ only in the substituent () attached to the a-carbon. The wide variation in these substituents (called side chains) is what gives proteins their great structural diversity and, as a consequence, their great functional diversity. Table 23.1 The Most ommon aturally ccurring Amino Acids The amino acids are shown in the form that predominates at physiological p (7.3). Average relative abundance Formula ame Abbreviations in proteins Aliphatic side chain amino acids 3 Glycine Gly G 7.5% 3 Alanine Ala A 9.0% Valine* Val V 6.9% 3 2 Leucine* Leu L 7.5% 2 3 Isoleucine* Ile I 4.6% 3 ydroxy-containing amino acids 2 3 erine er 7.1% Threonine* Thr T 6.0% 3 * Essential amino acids

ection 23.1 lassification and omenclature of Amino Acids 961 Table 23.1 (continued) Average relative abundance Formula ame Abbreviations in proteins ulfur-containing amino acids 2 3 ysteine ys 2.8% 2 2 3 Methionine* Met M 1.7% Acidic amino acids 2 Aspartate Asp D 5.5% (aspartic acid) 3 2 2 3 Glutamate Glu E 6.2% (glutamic acid) Amides of acidic amino acids 2 2 3 Asparagine Asn 4.4% 2 2 2 3 Glutamine Gln Q 3.9% Basic amino acids 3 2 2 2 2 Lysine* Lys K 7.0% 3 2 2 2 2 2 3 Arginine* Arg 4.7% Benzene-containing amino acids 2 3 Phenylalanine* Phe F 3.5% 2 Tyrosine Tyr Y 3.5% 3 eterocylic amino acids Η Η Proline Pro P 4.6% * Essential amino acids

962 APTE 23 Amino Acids, Peptides, and Proteins Table 23.1 (continued) Average relative abundance Formula ame Abbreviations in proteins eterocyclic amino acids (continued) 2 3 istidine* is 2.1% 2 Tryptophan* Trp W 1.1% 3 * Essential amino acids glycine leucine The amino acids are almost always called by their common names. ften, the name tells you something about the amino acid. For example, glycine got its name because of its sweet taste (glykos is Greek for sweet ), and valine, like valeric acid, has five carbon atoms. Asparagine was first found in asparagus, and tyrosine was isolated from cheese (tyros is Greek for cheese ). Dividing the amino acids into classes makes them easier to learn. The aliphatic side chain amino acids include glycine, the amino acid in which =, and four amino acids with alkyl side chains. Alanine is the amino acid with a methyl side chain, and valine has an isopropyl side chain. an you guess which amino acid leucine or isoleucine has an isobutyl side chain? If you gave the obvious answer, you guessed incorrectly. Isoleucine does not have an iso group; it is leucine that has an isobutyl substituent isoleucine has a sec-butyl substituent. Each of the amino acids has both a three-letter abbreviation (the first three letters of the name in most cases) and a single-letter abbreviation. Two amino acid side chains serine and threonine contain alcohol groups. erine is an -substituted alanine and threonine has a branched ethanol substituent. There are also two sulfur-containing amino acids: ysteine is an -substituted alanine and methionine has a 2-methylthioethyl substituent. There are two acidic amino acids (amino acids with two carboxylic acid groups): aspartate and glutamate. Aspartate is a carboxy-substituted alanine and glutamate has one more methylene group than aspartate. (If their carboxyl groups are protonated, they are called aspartic acid and glutamic acid, respectively.) Two amino acids asparagine and glutamine are amides of the acidic amino acids; asparagine is the amide of aspartate and glutamine is the amide of glutamate. otice that the obvious one-letter abbreviations cannot be used for these four amino acids because A and G are used for alanine and glycine. Aspartic acid and glutamic acid are abbreviated D and E, and asparagine and glutamine are abbreviated and Q. There are two basic amino acids (amino acids with two basic nitrogen-containing groups): lysine and arginine. Lysine has an P-amino group and arginine has a d-guanidino group. At physiological p, these groups are protonated. The P and d can remind you how many methylene groups each amino acid has. 3 2 2 2 2 2 2 2 2 2 an -amino group lysine 3 3 a -guanidino group arginine

Two amino acids phenylalanine and tyrosine contain benzene rings. As its name indicates, phenylalanine is phenyl-substituted alanine. Tyrosine is phenylalanine with a para-hydroxy substituent. Proline, histidine, and tryptophan are heterocyclic amino acids. Proline has its nitrogen incorporated into a five-membered ring it is the only amino acid that contains a secondary amino group. istidine is an imidazole-substituted alanine. Imidazole is an aromatic compound because it is cyclic and planar and has three pairs of delocalized p electrons (ection 21.11). The pk a of a protonated imidazole ring is 6.0, so the ring will be protonated in acidic solutions and nonprotonated in basic solutions (ection 23.3). ection 23.1 lassification and omenclature of Amino Acids 963 protonated imidazole imidazole aspartate Tryptophan is an indole-substituted alanine (ection 21.11). Like imidazole, indole is an aromatic compound. Because the lone pair on the nitrogen atom of indole is needed for the compound s aromaticity, indole is a very weak base. (The pk a of protonated indole is -2.4.) Therefore, the ring nitrogen in tryptophan is never protonated under physiological conditions. Ten amino acids are essential amino acids. We humans must obtain these 10 essential amino acids from our diets because we either cannot synthesize them at all or cannot synthesize them in adequate amounts. For example, we must have a dietary source of phenylalanine because we cannot synthesize benzene rings. owever, we do not need tyrosine in our diets, because we can synthesize the necessary amounts from phenylalanine. The essential amino acids are denoted by red asterisks (*) in Table 23.1. Although humans can synthesize arginine, it is needed for growth in greater amounts than can be synthesized. o arginine is an essential amino acid for children, but a nonessential amino acid for adults. ot all proteins contain the same amino acids. Bean protein is deficient in methionine, for example, and wheat protein is deficient in lysine. They are incomplete proteins: They contain too little of one or more essential amino acids to support growth. Therefore, a balanced diet must contain proteins from different sources. Dietary protein is hydrolyzed in the body to individual amino acids. ome of these amino acids are used to synthesize proteins needed by the body, some are broken down further to supply energy to the body, and some are used as starting materials for the synthesis of nonprotein compounds the body needs, such as adrenaline, thyroxine, and melanin (ection 25.6). PBLEM 1 a. Explain why, when the imidazole ring of histidine is protonated, the double-bonded nitrogen is the nitrogen that accepts the proton. lysine indole 2 2 2 2 3 b. Explain why, when the guanidino group of arginine is protonated, the double-bonded nitrogen is the nitrogen that accepts the proton. 2 2 2 2 2 2 2 2 2 2 Tutorial: Basic nitrogens in histidine and arginine 2 3

964 APTE 23 Amino Acids, Peptides, and Proteins 23.2 onfiguration of Amino Acids alanine an amino acid The a-carbon of all the naturally occurring amino acids except glycine is an asymmetric carbon. Therefore, 19 of the 20 amino acids listed in Table 23.1 can exist as enantiomers. The D and L notation used for monosaccharides (ection 22.2) is also used for amino acids. The D and L isomers of monosaccharides and amino acids are defined the same way. Thus, an amino acid drawn in a Fischer projection with the carboxyl group on the top and the group on the bottom of the vertical axis is a D-amino acid if the amino group is on the right and an L-amino acid if the amino group is on the left. Unlike monosaccharides, where the D isomer is the one found in nature, most amino acids found in nature have the L configuration. To date, D-amino acid residues have been found only in a few peptide antibiotics and in some small peptides attached to the cell walls of bacteria. 2 D-glyceraldehyde 3 D-amino acid 2 L-glyceraldehyde 3 L-amino acid Why D-sugars and L-amino acids? While it makes no difference which isomer nature selected to be synthesized, it is important that the same isomer be synthesized by all organisms. For example, if mammals ended up having L-amino acids, then L-amino acids would need to be the isomers synthesized by the organisms upon which mammals depend for food. AMI AID AD DIEAE The hamorro people of Guam have a high incidence of a syndrome that resembles amyotrophic lateral sclerosis (AL) with elements of Parkinson s disease and dementia. This syndrome developed during World War II when, as a result of food shortages, the tribe ate large quantities of ycas circinalis seeds. These seeds contain b-methylamino- L-alanine, an amino acid that binds to glutamate receptors. When monkeys are given b-methylamino-l-alanine, they develop some of the features of this syndrome. There is hope that, by studying the mechanism of action of b-methylamino- L-alanine, we may gain an understanding of how AL and Parkinson s disease arise. PBLEM 2 a. Which isomer ()-alanine or ()-alanine is D-alanine? b. Which isomer ()-aspartate or ()-aspartate is D-aspartate? c. an a general statement be made relating and to D and L? PBLEM 3 Which amino acids in Table 23.1 have more than one asymmetric carbon?

ection 23.3 Acid Base Properties of Amino Acids 965 23.3 Acid Base Properties of Amino Acids Every amino acid has a carboxyl group and an amino group, and each group can exist in an acidic form or a basic form, depending on the p of the solution in which the amino acid is dissolved. The carboxyl groups of the amino acids have pk a values of approximately 2, and the protonated amino groups have pk a values near 9 (Table 23.2). Both groups, therefore, will be in their acidic forms in a very acidic solution (p ' 0). At p = 7, the p of the solution is greater than the pk a of the carboxyl group, but less than the pk a of the protonated amino group. The carboxyl group, therefore, will be in its basic form and the amino group will be in its acidic form. In a strongly basic solution (p ' 11), both groups will be in their basic forms. ecall from the enderson asselbalch equation (ection 1.20) that the acidic form predominates if the p of the solution is less than the pk a of the compound and the basic form predominates if the p of the solution is greater than the pk a of the compound. 3 3 2 p = 0 a zwitterion p = 11 p = 7 otice that an amino acid can never exist as an uncharged compound, regardless of the p of the solution. To be uncharged, an amino acid would have to lose a proton from an 3 group with a pk a of about 9 before it would lose a proton from a group with a pk a of about 2. This clearly is impossible: A weak acid cannot be more acidic than a strong acid. Therefore, at physiological p (7.3) an amino acid exists as a dipolar ion, called a zwitterion. A zwitterion is a compound that has a negative charge Table 23.2 The pk a Values of Amino Acids pk a pk a pk a Amino acid A- A- side chain 3 Alanine 2.34 9.69 Arginine 2.17 9.04 12.48 Asparagine 2.02 8.84 Aspartic acid 2.09 9.82 3.86 ysteine 1.92 10.46 8.35 Glutamic acid 2.19 9.67 4.25 Glutamine 2.17 9.13 Glycine 2.34 9.60 istidine 1.82 9.17 6.04 Isoleucine 2.36 9.68 Leucine 2.36 9.60 Lysine 2.18 8.95 10.79 Methionine 2.28 9.21 Phenylalanine 2.16 9.18 Proline 1.99 10.60 erine 2.21 9.15 Threonine 2.63 9.10 Tryptophan 2.38 9.39 Tyrosine 2.20 9.11 10.07 Valine 2.32 9.62

966 APTE 23 Amino Acids, Peptides, and Proteins on one atom and a positive charge on a nonadjacent atom. (The name comes from zwitter, German for hermaphrodite or hybrid. ) A few amino acids have side chains with ionizable hydrogens (Table 23.2). The protonated imidazole side chain of histidine, for example, has a pk a of 6.04. istidine, therefore, can exist in four different forms, and the form that predominates depends on the p of the solution. 2 2 2 2 3 3 3 p = 0 p = 4 p = 8 p = 12 histidine 2 PBLEM 4 Why are the carboxylic acid groups of the amino acids so much more acidic (pk a ' 2) than a carboxylic acid such as acetic acid (pk a = 4.76)? PBLEM 5 LVED Draw the form in which each of the following amino acids predominantly exists at physiological p (7.3): a. aspartic acid c. glutamine e. arginine b. histidine d. lysine f. tyrosine LUTI T 5a Both carboxyl groups are in their basic forms because the p is greater than their pk a s. The protonated amino group is in its acidic form because the p is less than its pk a. 2 3 PBLEM 6 Draw the form in which glutamic acid predominantly exists in a solution with the following p: a. p = 0 b. p = 3 c. p = 6 d. p = 11 PBLEM 7 a. Why is the pk a of the glutamic acid side chain greater than the pk a of the aspartic acid side chain? b. Why is the pk a of the arginine side chain greater than the pk a of the lysine side chain? 23.4 The Isoelectric Point The isoelectric point (pi) of an amino acid is the p at which it has no net charge. In other words, it is the p at which the amount of positive charge on an amino acid exactly balances the amount of negative charge: pi (isoelectric point) p at which there is no net charge

The pi of an amino acid that does not have an ionizable side chain such as alanine is midway between its two pk a values. This is because at p = 2.34, half the molecules have a negatively charged carboxyl group and half have an uncharged carboxyl group, and at p = 9.69, half the molecules have a positively charged amino group and half have an uncharged amino group. As the p increases from 2.34, the carboxyl group of more molecules becomes negatively charged; as the p decreases from 9.69, the amino group of more molecules becomes positively charged. Therefore, at the average of the two pk a values, the number of negatively charged groups equals the number of positively charged groups. ection 23.4 The Isoelectric Point 967 pi pk a = 2.34 3 alanine pk a = 9.69 2.34 9.69 12.03 = = = 6.02 2 2 ecall from the enderson asselbalch equation that when p pk a, half the group is in its acidic form and half is in its basic form (ection 1.20). An amino acid will be positively charged if the p of the solution is less than the pi of the amino acid and will be negatively charged if the p of the solution is greater than the pi of the amino acid. The pi of an amino acid that has an ionizable side chain is the average of the pk a values of the similarly ionizing groups (a positively charged group ionizing to an uncharged group or an uncharged group ionizing to a negatively charged group). For example, the pi of lysine is the average of the pk a values of the two groups that are positively charged in their acidic form and uncharged in their basic form. The pi of glutamate, on the other hand, is the average of the pk a values of the two groups that are uncharged in their acidic form and negatively charged in their basic form. pk a = 10.79 3 2 2 2 2 pk a = 2.18 pk a = 2.19 2 2 pk 3 a = 4.25 3 pk a = 8.95 lysine glutamic acid pka = 9.67 pi 8.95 10.79 19.74 = = = 9.87 2 2 pi 2.19 4.25 6.44 = = = 3.22 2 2 PBLEM 8 Explain why the pi of lysine is the average of the pk a values of its two protonated amino groups. PBLEM 9 alculate the pi of each of the following amino acids: a. asparagine b. arginine c. serine PBLEM 10 a. Which amino acid has the lowest pi value? b. Which amino acid has the highest pi value? c. Which amino acid has the greatest amount of negative charge at p = 6.20? d. Which amino acid glycine or methionine has a greater negative charge at p = 6.20? PBLEM 11 Explain why the pi values of tyrosine and cysteine cannot be determined by the method just described.

968 APTE 23 Amino Acids, Peptides, and Proteins 23.5 eparation of Amino Acids Tutorial: Electrophoresis and pi Electrophoresis A mixture of amino acids can be separated by several different techniques. Electrophoresis separates amino acids on the basis of their pi values. A few drops of a solution of an amino acid mixture are applied to the middle of a piece of filter paper or to a gel. When the paper or the gel is placed in a buffered solution between two electrodes and an electric field is applied, an amino acid with a pi greater than the p of the solution will have an overall positive charge and will migrate toward the cathode (the negative electrode). The farther the amino acid s pi is from the p of the buffer, the more positive the amino acid will be and the farther it will migrate toward the cathode in a given amount of time. An amino acid with a pi less than the p of the buffer will have an overall negative charge and will migrate toward the anode (the positive electrode). If two molecules have the same charge, the larger one will move more slowly during electrophoresis because the same charge has to move a greater mass. ince amino acids are colorless, how can we detect that they have been separated? When amino acids are heated with ninhydrin, they form a colored product. After electrophoretic separation of the amino acids, the filter paper is sprayed with ninhydrin and dried in a warm oven. Most amino acids form a purple product. The number of different kinds of amino acids in the mixture is determined by the number of colored spots on the filter paper (Figure 23.1). The individual amino acids are identified by their location on the paper compared with a standard. cathode anode 2 2 3 2 2 2 2 3 3 3 arginine pl = 10.76 alanine pl = 6.02 Figure 23.1 Arginine, alanine, and aspartic acid separated by electrophoresis at p = 5. aspartate pl = 2.98 The mechanism for formation of the colored product is as shown, omitting the mechanisms for the steps involving dehydration, imine formation, and imine hydrolysis. (These mechanisms are shown in ections 18.6 and 18.7.) mechanism for the reaction of an amino acid with ninhydrin to form a colored product ninhydrin 2 2 an amino acid 2 2 2 2

ection 23.5 eparation of Amino Acids 969 2 purple-colored product 2 Paper hromatography and Thin-Layer hromatography Paper chromatography once played an important role in biochemical analysis because it provided a method for separating amino acids using very simple equipment. Although more modern techniques are now more commonly used, we will describe the principles behind paper chromatography because many of the same principles are employed in modern separation techniques. The technique of paper chromatography separates amino acids on the basis of polarity. A few drops of a solution of an amino acid mixture are applied to the bottom of a strip of filter paper. The edge of the paper is then placed in a solvent (typically a mixture of water, acetic acid, and butanol). The solvent moves up the paper by capillary action, carrying the amino acids with it. Depending on their polarities, the amino acids have different affinities for the mobile (solvent) and stationary (paper) phases and therefore travel up the paper at different rates. The more polar the amino acid, the more strongly it is adsorbed onto the relatively polar paper. The less polar amino acids travel up the paper more rapidly, since they have a greater affinity for the mobile phase. Therefore, when the paper is developed with ninhydrin, the colored spot closest to the origin is the most polar amino acid and the spot farthest away from the origin is the least polar amino acid (Figure 23.2). hromatography origin Leu Ala Glu least polar amino acid most polar amino acid > Figure 23.2 eparation of glutamate, alanine, and leucine by paper chromatography. The most polar amino acids are those with charged side chains, the next most polar are those with side chains that can form hydrogen bonds, and the least polar are those with hydrocarbon side chains. For amino acids with hydrocarbon side chains, the larger the alkyl group, the less polar the amino acid. In other words, leucine is less polar than valine. Paper chromatography has largely been replaced by thin-layer chromatography (TL). imilar to paper chromatography, TL differs from it in that TL uses a plate with a coating of solid material instead of filter paper. The physical property on which the separation is based depends on the solid material and the solvent chosen for the mobile phase. Movie: olumn chromatography PBLEM 12 A mixture of seven amino acids (glycine, glutamate, leucine, lysine, alanine, isoleucine, and aspartate) is separated by TL. Explain why only six spots show up when the chromatographic plate is sprayed with ninhydrin and heated.

970 APTE 23 Amino Acids, Peptides, and Proteins ations bind most strongly to cation-exchange resins. Anions bind most strongly to anion-exchange resins. Ion-Exchange hromatography Electrophoresis and thin-layer chromatography are analytical separations small amounts of amino acids are separated for analysis. Preparative separation, in which larger amounts of amino acids are separated for use in subsequent processes, can be achieved using ion-exchange chromatography. This technique employs a column packed with an insoluble resin. A solution of a mixture of amino acids is loaded onto the top of the column and eluted with a buffer. The amino acids separate because they flow through the column at different rates, as explained below. The resin is a chemically inert material with charged side chains. ne commonly used resin is a copolymer of styrene and divinylbenzene with negatively charged sulfonic acid groups on some of the benzene rings (Figure 23.3). If a mixture of lysine and glutamate in a solution with a p of 6 were loaded onto the column, glutamate would travel down the column rapidly because its negatively charged side chain would be repelled by the negatively charged sulfonic acid groups of the resin. The positively charged side chain of lysine, on the other hand, would cause that amino acid to be retained on the column. This kind of resin is called a cation-exchange - resin because it exchanges the a counterions of the 3 groups for the positively charged species that are added to the column. In addition, the relatively nonpolar nature of the column causes it to retain nonpolar amino acids longer than polar amino acids. esins with positively charged groups are called anion-exchange resins because they impede the flow of anions by exchanging their negatively charged counterions for negatively charged species that are added to the column. A common anionexchange resin (Dowex 1) has groups in place of the - 3 a 2 ( ) 3 l - groups in Figure 23.3. Figure 23.3 A section of a cation-exchange resin. This particular resin is called Dowex 50. 2 3 a 3 a 3 a 2 2 2 2 2 2 2 2 2 2 2 3 a 3 a An amino acid analyzer is an instrument that automates ion-exchange chromatography. When a solution of an amino acid mixture passes through the column of an amino acid analyzer containing a cation-exchange resin, the amino acids move through the column at different rates, depending on their overall charge. The solution leaving the column is collected in fractions, which are collected often enough that a different amino acid ends up in each fraction (Figure 23.4). If ninhydrin is added to each of the fractions, the concentration of the amino acid in each fraction can be WATE FTEE: EXAMPLE F ATI- EXAGE MATGAPY Water softeners contain a column with a cation-exchange resin that has been flushed with concentrated sodium chloride. In ection 17.13, we saw that the presence of calcium and magnesium ions in water is what causes the water to be hard. When water passes through the column, the resin binds magnesium and calcium ions more tightly than it binds sodium ions. In this way, the water softener removes magnesium and calcium ions from water, replacing them with sodium ions. The resin must be recharged from time to time by flushing it with concentrated sodium chloride to replace the bound magnesium and calcium ions with sodium ions.

ection 23.5 eparation of Amino Acids 971 > Figure 23.4 eparation of amino acids by ion-exchange chromatography. Fractions sequentially collected determined by the amount of absorption at 570 nm because the colored compound formed by the reaction of an amino acid with ninhydrin has a l max of 570 (ection 8.11). In this way, the identity and the relative amount of each amino acid can be determined (Figure 23.5). Absorbance p 3.3 buffer Asp Thr er Glu Gly Pro Ala p 4.3 buffer Met Ile Leu Val Tyr Phe Lys p 5.3 buffer > Figure 23.5 A typical chromatogram obtained is 3 Arg from the separation of a mixture of amino acids using an automated amino acid analyzer. 40 80 120 160 200 240 280 320 330 370 410 450 490 50 90 130 Effluent (ml) PBLEM 13 Why are buffer solutions of increasingly higher p used to elute the column that generates the chromatogram shown in Figure 23.5? PBLEM 14 Explain the order of elution (with a buffer of p 4) of each of the following pairs of amino acids on a column packed with Dowex 50 (Figure 23.3): a. aspartate before serine c. valine before leucine b. glycine before alanine d. tyrosine before phenylalanine PBLEM 15 In what order would the following amino acids be eluted with a buffer of p 4 from a column containing an anion-exchange resin? histidine, serine, aspartate, valine

972 APTE 23 Amino Acids, Peptides, and Proteins 23.6 esolution of acemic Mixtures of Amino Acids hemists do not have to rely on nature to produce amino acids; they can synthesize them in the laboratory, using a variety of methods. ne of the oldest methods replaces an a-hydrogen of a carboxylic acid with a bromine in a ell Volhard Zelinski reaction (ection 19.5). The resulting a-bromocarboxylic acid then undergoes an 2 reaction with ammonia to form the amino acid (ection 10.4). 2 a carboxylic acid 1. Br 2, PBr 3 2. 3 Br excess 3 3 an amino acid 4 Br PBLEM 16 Why is excess ammonia used in the preceding reaction? When amino acids are synthesized in nature, only the L-enantiomer is formed (ection 5.20). owever, when amino acids are synthesized in the laboratory, the product is usually a racemic mixture of D and L enantiomers. If only one isomer is desired, the enantiomers must be separated. They can be separated by means of an enzyme-catalyzed reaction. Because an enzyme is chiral, it will react at a different rate with each of the enantiomers (ection 5.20). For example, pig kidney aminoacylase is an enzyme that catalyzes the hydrolysis of -acetyl-l-amino acids, but not -acetyl- D-amino acids. Therefore, if the racemic amino acid is converted into a pair of -acetylamino acids and the -acetylated mixture is hydrolyzed with pig kidney aminoacylase, the products will be the L-amino acid and -acetyl-d-amino acid, which are easily separated. Because the resolution (separation) of the enantiomers depends on the difference in the rates of reaction of the enzyme with the two -acetylated compounds, this technique is known as a kinetic resolution. 2 D-amino acid L-amino acid pig kidney 2 2 -acetyl-d-amino acid L-amino acid -acetyl-l-amino acid -acetyl-d-amino acid PBLEM 17 Pig liver esterase is an enzyme that catalyzes the hydrolysis of esters. It hydrolyzes esters of L-amino acids more rapidly than esters of D-amino acids. ow can this enzyme be used to separate a racemic mixture of amino acids? PBLEM 18 Amino acids can be synthesized by reductive amination of a-keto acids (ection 21.8). excess ammonia 2 /aney i 3

Biological organisms can also convert a-keto acids into amino acids, but because 2 and metal catalysts are not available to the cell, they do so by a different mechanism (ection 25.6.) a. What amino acid is obtained from the reductive amination of each of the following metabolic intermediates in the cell? 2 2 2 pyruvic acid oxaloacetic acid -ketoglutaric acid b. What amino acids are obtained from the same metabolic intermediates when they are synthesized in the laboratory? ection 23.7 Peptide Bonds and Disulfide Bonds 973 23.7 Peptide Bonds and Disulfide Bonds Peptide bonds and disulfide bonds are the only covalent bonds that hold amino acid residues together in a peptide or a protein. Peptide Bonds The amide bonds that link amino acid residues are called peptide bonds. By convention, peptides and proteins are written with the free amino group (the -terminal amino acid) on the left and the free carboxyl group (the -terminal amino acid) on the right. 3 3 3 the -terminal amino acid 3 peptide bonds a tripeptide 2 2 the -terminal amino acid When the identities of the amino acids in a peptide are known but their sequence is not known, the amino acids are written separated by commas. When the sequence of amino acids is known, the amino acids are written separated by hyphens. In the following pentapeptide shown on the right, valine is the -terminal amino acid and histidine is the -terminal amino acid. The amino acids are numbered starting with the -terminal end. The glutamate residue is referred to as Glu 4 because it is the fourth amino acid from the -terminal end. In naming the peptide, adjective names (ending in yl ) are used for all the amino acids except the -terminal amino acid. Thus, this pentapeptide is named valylcysteylalanylglutamylhistidine. Glu, ys, is, Val, Ala the pentapeptide contains the indicated amino acids, but their sequence is not known Val-ys-Ala-Glu-is the amino acids in the pentapeptide have the indicated sequence A peptide bond has about 40% double-bond character because of electron delocalization. teric hindrance causes the trans configuration to be more stable than the

974 APTE 23 Amino Acids, Peptides, and Proteins cis configuration, so the a-carbons of adjacent amino acids are trans to each other (ection 4.11). -carbon -carbon trans configuration Free rotation about the peptide bond is not possible because of its partial double-bond character. The carbon and nitrogen atoms of the peptide bond and the two atoms to which each is attached are held rigidly in a plane (Figure 23.6). This regional planarity affects the way a chain of amino acids can fold, so it has important implications for the three-dimensional shapes of peptides and proteins (ection 23.13). Figure 23.6 A segment of a polypeptide chain. The plane defined by each peptide bond is indicated. otice that the groups bonded to the a-carbons are on alternate sides of the peptide backbone. PBLEM 19 Draw a peptide bond in a cis configuration. Disulfide Bonds When thiols are oxidized under mild conditions, they form disulfides. A disulfide is a compound with an bond. 2 a thiol mild oxidation a disulfide An oxidizing agent commonly used for this reaction is Br 2 (or I 2 ) in a basic solution. mechanism for oxidation of a thiol to a disulfide 2 Br Br Br Br Br Because thiols can be oxidized to disulfides, disulfides can be reduced to thiols. ysteine is an amino acid that contains a thiol group. Two cysteine molecules therefore can be oxidized to a disulfide. This disulfide is called cystine. 2 2 a disulfide reduction mild oxidation 2 a thiol 2 2 3 3 3 cysteine cystine Two cysteine residues in a protein can be oxidized to a disulfide. This is known as a disulfide bridge. Disulfide bridges are the only covalent bonds that can form between nonadjacent amino acids. They contribute to the overall shape of a protein by holding the cysteine residues in close proximity, as shown in Figure 23.7.

ection 23.7 Peptide Bonds and Disulfide Bonds 975 > Figure 23.7 Disulfide bridges cross-linking portions of a peptide. oxidation reduction polypeptide disulfide bridges cross-linking portions of a polypeptide Insulin, a hormone secreted by the pancreas, controls the level of glucose in the blood by regulating glucose metabolism. Insulin is a polypeptide with two peptide chains. The short chain (the A-chain) contains 21 amino acids and the long chain (the B-chain) contains 30 amino acids. The two chains are held together by two disulfide bridges. These are interchain disulfide bridges (between the A- and B-chains). Insulin also has an intrachain disulfide bridge (within the A-chain). an intrachain disulfide bridge A-chain Gly Ile Val Glu Glnys ys Thr er Ile ys er Leu Tyr Gln Leu Glu Asn Tyr ys Asn interchain disulfide bridges B-chain Phe Val Asn Gln is Leu ys Gly er is Leu Val Glu Ala Leu Tyr Leu Val ys Gly GluArg Gly Phe Phe Tyr Thr Pro Lys Ala insulin AI: TAIGT ULY? air is made up of a protein known as keratin. Keratin contains an unusually large number of cysteine residues (about 8% of the amino acids), which give it many disulfide bridges to maintain its three-dimensional structure. People can alter the structure of their hair (if they feel that it is either too straight or too curly) by changing the location of these disulfide bridges. This is accomplished by first applying a reducing agent to the hair to reduce all the disulfide bridges in the protein strands. Then the hair is given the desired shape (using curlers to curl it or combing it straight to uncurl it), and an oxidizing agent is applied that forms new disulfide bridges. The new disulfide bridges maintain the hair s new shape. When this treatment is applied to straight hair, it is called a permanent. When it is applied to curly hair, it is called hair straightening. curly hair straight hair

976 APTE 23 Amino Acids, Peptides, and Proteins PBLEM 20 a. ow many different octapeptides can be made from the 20 naturally occurring amino acids? b. ow many different proteins containing 100 amino acids can be made from the 20 naturally occurring amino acids? PBLEM 21 Which bonds in the backbone of a peptide can rotate freely? 23.8 ome Interesting Peptides xytocin was the first small peptide to be synthesized. Its synthesis was achieved in 1953 by Vincent du Vigneaud (1901 1978), who later synthesized vasopressin. Du Vigneaud was born in hicago and was a professor at George Washington University Medical chool and later at ornell University Medical ollege. For synthesizing these nonapeptides, he received the obel Prize in chemistry in 1955. Enkephalins are pentapeptides that are synthesized by the body to control pain. They decrease the body s sensitivity to pain by binding to receptors in certain brain cells. Part of the three-dimensional structures of enkephalins must be similar to those of morphine and painkillers such as Demerol because they bind to the same receptors (ections 30.3 and 30.6). Tyr-Gly-Gly-Phe-Leu leucine enkephalin Bradykinin, vasopressin, and oxytocin are peptide hormones. They are all nonapeptides. Bradykinin inhibits the inflammation of tissues. Vasopressin controls blood pressure by regulating the contraction of smooth muscle. It is also an antidiuretic. xytocin induces labor in pregnant women and stimulates milk production in nursing mothers. Vasopressin and oxytocin both have an intrachain disulfide bond, and their -terminal amino acids contain amide rather than carboxyl groups. otice that the -terminal amide group is indicated by writing 2 after the name of the -terminal amino acid. In spite of their very different physiological effects, vasopressin and oxytocin differ only by two amino acids. bradykinin Tyr-Gly-Gly-Phe-Met methionine enkephalin Arg-Pro-Pro-Gly-Phe-er-Pro-Phe-Arg vasopressin ys-tyr-phe-gln-asn-ys-pro-arg-gly- 2 oxytocin ys-tyr-ile-gln-asn-ys-pro-leu-gly- 2 L-Val L-Pro L-rn L-Phe L-Leu L-Leu D-Phe D-rn L-Pro L-Val gramicidin 3 2 2 2 3 ornithine Gramicidin is an antibiotic produced by a strain of bacteria. It is a cyclic decapeptide. otice that it contains the amino acids L-ornithine (L-rn), D-ornithine (D-rn), and also D-phenylalanine. rnithine is not listed in Table 23.1 because it occurs rarely in nature. rnithine resembles lysine, but has one less methylene group in its side chain. The synthetic sweetener aspartame, or utraweet (ection 22.21), is the methyl ester of a dipeptide of L-aspartate and L-phenylalanine. Aspartame is about 200 times sweeter than sucrose. The ethyl ester of the same dipeptide is not sweet. If a D-amino acid is substituted for either of the L-amino acids of aspartame, the resulting dipeptide is bitter rather than sweet. 3 2 2 aspartame utraweet

ection 23.9 trategy of Peptide Bond ynthesis: -Protection and -Activation 977 Glutathione is a tripeptide of glutamate, cysteine, and glycine. Its function is to destroy harmful oxidizing agents in the body. xidizing agents are thought to be responsible for some of the effects of aging and are believed to play a role in cancer (ection 9.8). Glutathione removes oxidizing agents by reducing them. onsequently, glutathione is oxidized, forming a disulfide bond between two glutathione molecules. An enzyme subsequently reduces the disulfide bond, allowing glutathione to react with more oxidizing agents. 2 3 2 2 2 glutathione reducing agent 2 oxidizing agent 3 2 2 2 3-D Molecules: Glutathione; xidized glutathione 2 2 3 2 2 2 oxidized glutathione PBLEM 22 What is unusual about glutathione s structure? (If you can t answer this question, draw the structure you would expect for a tripeptide of glutamate, cysteine, and glycine, and compare your structure with the structure of glutathione.) 23.9 trategy of Peptide Bond ynthesis: -Protection and -Activation Because amino acids have two functional groups, a problem arises when one attempts to make a particular peptide bond. For example, suppose you wanted to make the dipeptide Gly-Ala. That dipeptide is only one of four possible dipeptides that could be formed from alanine and glycine. 3 2 3 3 2 2 3 2 Gly-Ala Ala-Ala Gly-Gly Ala-Gly If the amino group of the amino acid that is to be on the -terminal end (in this case, Gly) is protected, it will not be available to form a peptide bond. If the carboxyl group of this same amino acid is activated before the second amino acid is added, the amino group of the added amino acid (in this case, Ala) will react with the activated

978 APTE 23 Amino Acids, Peptides, and Proteins carboxyl group of glycine in preference to reacting with a nonactivated carboxyl group of another alanine molecule. protect glycine 2 2 activate alanine 2 peptide bond is formed between these groups The reagent that is most often used to protect the amino group of an amino acid is di-tert-butyl dicarbonate. Its popularity is due to the ease with which the protecting group can be removed when the need for protection is over. The protecting group is known by the acronym t-b (pronounced tee-boc). di-tert-butyl dicarbonate 2 2 glycine 2 3 2 -protected glycine arboxylic acids are generally activated by being converted into acyl chlorides (ection 17.20). Acyl chlorides, however, are so reactive that they can readily react with the substituents of some of the amino acids during peptide synthesis, creating unwanted products. The preferred method for activating the carboxyl group of an -protected amino acid is to convert it into an imidate using dicyclohexylcarbodiimide (D). (By now, you have probably noticed that biochemists are even more fond of acronyms than organic chemists are.) D activates a carboxyl group by putting a good leaving group on the carbonyl carbon. 2 proton transfer 2 -protected amino acid dicyclohexylcarbodiimide D 2 protected activated an imidate After the amino acid has its -terminal group protected and its -terminal group activated, the second amino acid is added to form the new peptide bond. The bond of the tetrahedral intermediate is easily broken (the activated group is a good leaving

ection 23.9 trategy of Peptide Bond ynthesis: -Protection and -Activation 979 group) because the bonding electrons are delocalized, forming dicyclohexylurea, a stable diamide. [ecall that the weaker (more stable) the base, the better it is as a leaving group; see ection 17.5.] B 2 2 amino acid 2 B 3 tetrahedral intermediate 2 new peptide bond dicyclohexylurea a diamide Amino acids can be added to the growing -terminal end by repeating these two steps: activating the carboxyl group of the -terminal amino acid of the peptide by treating it with D and then adding a new amino acid. 3 2 -protected dipeptide 1. D 2. 2 2 -protected tripeptide When the desired number of amino acids has been added to the chain, the protecting group on the -terminal amino acid is removed. t-b is an ideal protecting group because it can be removed by washing with trifluoroacetic acid and methylene chloride, reagents that will not break any other covalent bonds. The protecting group is removed by an elimination reaction, forming isobutylene and carbon dioxide. Because these products are gases, they escape, driving the reaction to completion. B 2 2 -protected tripeptide F 3 2 3 2 tripeptide F 3 2 l 2 B 2 2

980 APTE 23 Amino Acids, Peptides, and Proteins Theoretically, one should be able to make as long a peptide as desired with this technique. eactions do not produce 100% yields, however, and the yields are further decreased during the purification process. After each step of the synthesis, the peptide must be purified to prevent subsequent unwanted reactions with leftover reagents. Assuming that each amino acid can be added to the growing end of the peptide chain in an 80% yield (a relatively high yield, as you can probably appreciate from your own experience in the laboratory), the overall yield of a nonapeptide such as bradykinin would be only 17%. It is clear that large polypeptides could never be synthesized in this way. umber of amino acids 2 3 4 5 6 7 8 9 verall yield 80% 64% 51% 41% 33% 26% 21% 17% PBLEM 23 What dipeptides would be formed by heating a mixture of valine and -protected leucine? PBLEM 24 uppose you are trying to synthesize the dipeptide Val-er. ompare the product that would be obtained if the carboxyl group of -protected valine were activated with thionyl chloride with the product that would be obtained if the carboxyl group were activated with D. PBLEM 25 how the steps in the synthesis of the tetrapeptide Leu-Phe-Lys-Val. PBLEM 26 a. alculate the overall yield of bradykinin if the yield for the addition of each amino acid to the chain is 70%. b. What would be the overall yield of a peptide containing 15 amino acid residues if the yield for the incorporation of each is 80%? 23.10 Automated Peptide ynthesis. Bruce Merrifield was born in 1921 and received a B.. and a Ph.D. from the University of alifornia, Los Angeles. e is a professor of chemistry at ockefeller University. Merrifield received the 1984 obel Prize in chemistry for developing automated solid-phase peptide synthesis. In addition to producing low overall yields, the method of peptide synthesis described in ection 23.9 is extremely time-consuming because the product must be purified at each step of the synthesis. In 1969, Bruce Merrifield described a method that revolutionized the synthesis of peptides because it provided a much faster way to produce peptides in much higher yields. Furthermore, because it is automated, the synthesis requires fewer hours of direct attention. Using this technique, bradykinin was synthesized with an 85% yield in 27 hours. ubsequent refinements in the technique now allow a reasonable yield of a peptide containing 100 amino acids to be synthesized in four days. In the Merrifield method, the -terminal amino acid is covalently attached to a solid support contained in a column. Each -terminal blocked amino acid is added one at a time, along with other needed reagents, so the protein is synthesized from the -terminal end to the -terminal end. otice that this is opposite to the way proteins are synthesized in nature (from the -terminal end to the -terminal end; ection 27.13). Because it uses a solid support and is automated, Merrifield s method of protein synthesis is called automated solid-phase peptide synthesis. Merrifield automated solid-phase synthesis of a tripeptide l 2 -protected amino acid resin

ection 23.10 Automated Peptide ynthesis 981 -protected amino acid 2 D 2 2 2 2 -protected and -activated amino acid D F 3 2 l 2 Tutorial: Merrifield automated solid-phase synthesis 2 2 2 2 2 F 3 2 l 2 -protected amino acid D -protected and -activated amino acid 2 D F 3 2 l 2 2 2 2 2 F 3 2

982 APTE 23 Amino Acids, Peptides, and Proteins The solid support to which the -terminal amino acid is attached is a polystyrene resin similar to the one used in ion-exchange chromatography (ection 23.5), except that the benzene rings have chloromethyl substituents instead of sulfonic acid substituents. Before the -terminal amino acid is attached to the resin, its amino group is protected with t-b to prevent the amino group from reacting with the resin. The -terminal amino acid is attached to the resin by means of an 2 reaction its carboxyl group attacks a benzyl carbon of the resin, displacing a chloride ion (ection 10.4). After the -terminal amino acid is attached to the resin, the t-b protecting group is removed (ection 23.9). The next amino acid, with its amino group protected with t-b and its carboxyl group activated with D, is added to the column. A huge advantage of the Merrifield method of peptide synthesis is that the growing peptide can be purified by washing the column with an appropriate solvent after each step of the procedure. The impurities are washed out of the column because they are not attached to the solid support. ince the peptide is covalently attached to the resin, none of it is lost in the purification step, leading to high yields of purified product. After the required amino acids have been added one by one, the peptide can be removed from the resin by treatment with F under mild conditions that do not break the peptide bonds. Merrifield s technique is constantly being improved so that peptides can be made more rapidly and more efficiently. owever, it still cannot begin to compare with nature: A bacterial cell is able to synthesize a protein thousands of amino acids long in seconds and can simultaneously synthesize thousands of different proteins with no mistakes. ince the early 1980s, it has been possible to synthesize proteins by genetic engineering techniques. trands of DA can be introduced into bacterial cells, causing the cells to produce large amounts of a desired protein (ection 27.13). For example, mass quantities of human insulin are produced from genetically modified E. coli. Genetic engineering techniques also have been useful in synthesizing proteins that differ in one or a few amino acids from the natural protein. uch synthetic proteins have been used, for example, to learn how a change in a single amino acid affects the properties of a protein (ection 24.9). PBLEM 27 how the steps in the synthesis of the peptide in Problem 25, using Merrifield s method. 23.11 Protein tructure Protein molecules are described by several levels of structure. The primary structure of a protein is the sequence of amino acids in the chain and the location of all the disulfide bridges. The secondary structure describes the regular conformation assumed by segments of the protein s backbone. In other words, the secondary structure describes how local regions of the backbone fold. The tertiary structure describes the three-dimensional structure of the entire polypeptide. If a protein has more than one polypeptide chain, it has quaternary structure. The quaternary structure of a protein is the way the individual protein chains are arranged with respect to each other. Proteins can be divided roughly into two classes. Fibrous proteins contain long chains of polypeptides that occur in bundles. These proteins are insoluble in water. All the structural proteins described at the beginning of this chapter, such as keratin and collagen, are fibrous proteins. Globular proteins are soluble in water and tend to have roughly spherical shapes. Essentially all enzymes are globular proteins.