CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids H3N R (achiral of an c- amino acid PROBLEM 27.1 What is the absolute configuration(R or S )at the c carbon atom in each of the following L-amino acids? (a)H3N-+H CO2 (b)H3N SAMPLE SOLUTION (a) First identify the four groups attached directly to the stereogenic center, and rank them in order of decreasing sequence rule prece- dence For L-serine these groups are Next, translate the Fischer projection of L-serine to a three-dimensional represen- tation, and orient it so that the lowest ranked substituent at the stereogenic cen CH2OH In order of decreasing precedence the three highest ranked groups trace an anti clockwise path HOCH he absolute configuration of L-serine is S PRobLEM 27.2 Which of the amino acids in table 27.1 have more than one tereogenic center? Although all the chiral amino acids obtained from proteins have the L configura tion at their a carbon that should not be taken to mean that D-amino acids are unknown In fact, quite a number of D-amino acids occur naturally. D-Alanine, for example, is Back Forward Main MenuToc Study Guide ToC Student o MHHE Website
PROBLEM 27.1 What is the absolute configuration (R or S) at the carbon atom in each of the following L-amino acids? (a) (c) (b) SAMPLE SOLUTION (a) First identify the four groups attached directly to the stereogenic center, and rank them in order of decreasing sequence rule precedence. For L-serine these groups are Next, translate the Fischer projection of L-serine to a three-dimensional representation, and orient it so that the lowest ranked substituent at the stereogenic center is directed away from you. In order of decreasing precedence the three highest ranked groups trace an anticlockwise path. The absolute configuration of L-serine is S. PROBLEM 27.2 Which of the amino acids in Table 27.1 have more than one stereogenic center? Although all the chiral amino acids obtained from proteins have the L configuration at their carbon, that should not be taken to mean that D-amino acids are unknown. In fact, quite a number of D-amino acids occur naturally. D-Alanine, for example, is a HOCH2 CO2 NH3 H3N CO2 H CH2OH C HOCH2 H CO2 NH3 C NH3 HOCH2 CO2 H H3N± Highest ranked H Lowest ranked ±CO2 ±CH2OH H3N CO2 H CH2SH L-Cysteine H3N CO2 H CH2CH2SCH3 L-Methionine H3N CO2 H CH2OH L-Serine H3N CO2 H H Glycine (achiral) Fischer projection of an L-amino acid H3N CO2 H R C R H NH3 CO2 1056 CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
27.3 Acid-Base Behavior of amino acids constituent of bacterial cell walls. The point is that D-amino acids are not constituents of A new technique for dating archaeological samples called amino acid racemiza tion(AAr)is based on the stereochemistry of amino acids. Over time, the configuration at the a-carbon atom of a proteins amino acids is lost in a reaction that follows first- order kinetics. When the a carbon is the only stereogenic center, this process corresponds to racemization For an amino acid with two stereogenic centers changing the configu ration of the a carbon from L to D gives a diastereomer. In the case of isoleucine, for example, the diastereomer is an amino acid not normally present in proteins, called alloisoleucine H3C D-Alloisoleucine y measuring the L-isoleucine/D-alloisoleucine ratio in the protein isolated from the eggshells of an extinct Australian bird, a team of scientists recently determined that thi bird lived approximately 50,000 years ago. Radiocarbon (C)dating is not accurate for samples older than about 35, 000 years, so AAR is a useful addition to the tools avail able to paleontologists 27.3 ACID-BASE BEHAVIOR OF AMINO ACIDS The physical properties of a typical amino acid such as glycine suggest that it is a very polar substance, much more polar than would be expected on the basis of its formula- tion as H2NCH2CO2H. Glycine is a crystalline solid; it does not melt, but on being heated it eventually decomposes at 233C. It is very soluble in water but practically insoluble in nonpolar organic solvents. These properties are attributed to the fact that the stable form of glycine is a zwitterion, or inner salt. The zwitterion is also ofte referred to as a dipolar ion. H2NCH2C、 H3NCH2C、 an ion but a neutral m Zwitterionic form of glycine The equilibrium expressed by the preceding equation lies overwhelmingly to the side of the zwitterion Glycine, as well as other amino acids, is amphoteric, meaning it contains an acidic functional group and a basic functional group. The acidic functional group is the ammo- nium ion H3N-; the basic functional group is the carboxylate ion -CO2. How do we know this? Aside from its physical properties, the acid-base properties of glycine, as lustrated by the titration curve in Figure 27. 2, require it. In a strongly acidic medium the species present is H3NCH2CO2H. As the pH is raised, a proton is removed from this species Is the proton removed from the positively charged nitrogen or from the carboxyl group? We know what to expect for the relative acid strengths of RNH3 and RCO2H. A pical ammonium ion has pKa =9, and a typical carboxylic acid has pK a s 5. The Back Forward Main MenuToc Study Guide ToC Student o MHHE Website
constituent of bacterial cell walls. The point is that D-amino acids are not constituents of proteins. A new technique for dating archaeological samples called amino acid racemization (AAR) is based on the stereochemistry of amino acids. Over time, the configuration at the -carbon atom of a protein’s amino acids is lost in a reaction that follows firstorder kinetics. When the carbon is the only stereogenic center, this process corresponds to racemization. For an amino acid with two stereogenic centers, changing the configuration of the carbon from L to D gives a diastereomer. In the case of isoleucine, for example, the diastereomer is an amino acid not normally present in proteins, called alloisoleucine. By measuring the L-isoleucine/D-alloisoleucine ratio in the protein isolated from the eggshells of an extinct Australian bird, a team of scientists recently determined that this bird lived approximately 50,000 years ago. Radiocarbon (14C) dating is not accurate for samples older than about 35,000 years, so AAR is a useful addition to the tools available to paleontologists. 27.3 ACID–BASE BEHAVIOR OF AMINO ACIDS The physical properties of a typical amino acid such as glycine suggest that it is a very polar substance, much more polar than would be expected on the basis of its formulation as H2NCH2CO2H. Glycine is a crystalline solid; it does not melt, but on being heated it eventually decomposes at 233°C. It is very soluble in water but practically insoluble in nonpolar organic solvents. These properties are attributed to the fact that the stable form of glycine is a zwitterion, or inner salt. The equilibrium expressed by the preceding equation lies overwhelmingly to the side of the zwitterion. Glycine, as well as other amino acids, is amphoteric, meaning it contains an acidic functional group and a basic functional group. The acidic functional group is the ammonium ion ; the basic functional group is the carboxylate ion ±CO2 . How do we know this? Aside from its physical properties, the acid–base properties of glycine, as illustrated by the titration curve in Figure 27.2, require it. In a strongly acidic medium the species present is . As the pH is raised, a proton is removed from this species. Is the proton removed from the positively charged nitrogen or from the carboxyl group? We know what to expect for the relative acid strengths of and RCO2H. A typical ammonium ion has pKa 9, and a typical carboxylic acid has pKa 5. The RNH3 H3NCH2CO2H H3N± H2NCH2C O OH H3NCH2C O O Zwitterionic form of glycine L-Isoleucine CO2 CH2CH3 H3N H H3C H D-Alloisoleucine CO2 CH2CH3 H NH3 H3C H 27.3 Acid–Base Behavior of Amino Acids 1057 The zwitterion is also often referred to as a dipolar ion. Note, however, that it is not an ion, but a neutral molecule. Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
1058 CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids FIGURE 27.2 The titration curve of glycine. At pH values less than pkat, H3NCH2CO 2H is the major species present. At pH values between pKal and pKa2, the principal species is the zwitterion H3 NCH2CO2. The concentration of the zwitterion is a maximum at the isoelectric point pl. At pH values greater than pKa2, H2NCH2CO2 is the species present in greatest concentratio measured pka for the conjugate acid of glycine is 2.35, a value closer to that expected for deprotonation of the carboxyl group. As the ph is raised, a second deprotonation ep, corresponding to removal of a proton from nitrogen of the zwitterion, is observed The pKa associated with this step is 9.78, much like that of typical alkylammonium ions H3 NCh,C H3NCH2C HNCHC Species present Zwitterion; predominant Species pre strong a species in solutions near Thus, glycine is characterized by two pKa values: the one corresponding to the more acidic site is designated pKal, the one corresponding to the less acidic site is des- ignated pKa2. Table 27.2 lists pKal and pKa2 values for the a-amino acids that have neu- tral side chains, which are the first two groups of amino acids given in Table 27. 1. In all cases their pKa values are similar to those of glycine Table 27. 2 includes a column labeled pl, which gives isoelectric point values. The isoelectric point is the pH at which the amino acid bears no net charge; it corresponds to the ph at which the concentration of the zwitterion is a maximum. For the amino acids in Table 27. 2 this is the average of pKal and pka and lies slightly to the acid side Some amino acids including those listed in the last two sections of Table 27.1 have side chains that bear acidic or basic groups. As Table 27.3 indicates, these amino acids are characterized by three pka values. The"extra"pka value(it can be either pKa2 or pKa3)reflects the nature of the function present in the side chain. The isoelectric points of the amino acids in Table 27.3 are midway between the pKa values of the monocation and monoanion and are well removed from neutrality when the side chain bears a car boxyl group(aspartic acid, for example)or a basic amine function(lysine, for example). Back Forward Main MenuToc Study Guide ToC Student o MHHE Website
measured pKa for the conjugate acid of glycine is 2.35, a value closer to that expected for deprotonation of the carboxyl group. As the pH is raised, a second deprotonation step, corresponding to removal of a proton from nitrogen of the zwitterion, is observed. The pKa associated with this step is 9.78, much like that of typical alkylammonium ions. Thus, glycine is characterized by two pKa values: the one corresponding to the more acidic site is designated pKa1, the one corresponding to the less acidic site is designated pKa2. Table 27.2 lists pKa1 and pKa2 values for the -amino acids that have neutral side chains, which are the first two groups of amino acids given in Table 27.1. In all cases their pKa values are similar to those of glycine. Table 27.2 includes a column labeled pI, which gives isoelectric point values. The isoelectric point is the pH at which the amino acid bears no net charge; it corresponds to the pH at which the concentration of the zwitterion is a maximum. For the amino acids in Table 27.2 this is the average of pKa1 and pKa2 and lies slightly to the acid side of neutrality. Some amino acids, including those listed in the last two sections of Table 27.1, have side chains that bear acidic or basic groups. As Table 27.3 indicates, these amino acids are characterized by three pKa values. The “extra” pKa value (it can be either pKa2 or pKa3) reflects the nature of the function present in the side chain. The isoelectric points of the amino acids in Table 27.3 are midway between the pKa values of the monocation and monoanion and are well removed from neutrality when the side chain bears a carboxyl group (aspartic acid, for example) or a basic amine function (lysine, for example). H3NCH2C O O Zwitterion; predominant species in solutions near neutrality H2NCH2C O O Species present in strong base Species present in strong acid H3NCH2C O OH H H H H 1058 CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids Equivalents of base added 0.4 0.2 0.0 1.0 0.8 0.6 1.6 1.4 1.2 2.0 1.8 pH 2 4 6 8 10 12 pKa1 = 2.3 pKa2 = 9.8 pI FIGURE 27.2 The titration curve of glycine. At pH values less than pKa1, is the major species present. At pH values between pKa1 and pKa2, the principal species is the zwitterion . The concentration of the zwitterion is a maximum at the isoelectric point pI. At pH values greater than pKa2, H2NCH2CO2 is the species present in greatest concentration. H3NCH2CO2 H3NCH2CO2H Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
27.3 Acid-Base Behavior of amino acids TABLE 27.2 Acid-Base Properties of Amino Acids with Neutral Side Chains Amino acid pKa,* pKa?" Glycine 2.34 9.60 5.97 2.34 9.69 6.00 valine 2.32 9.62 5.96 2.36 9.60 5.98 Isoleucine 2.36 9.60 6.02 Methionine 2.28 9.21 5.74 Proline 10.60 6.30 1.83 9.13 5.48 Tryptophan 2.83 9.39 5.89 2.02 8.80 5.41 Glutamine 2.17 9.13 5.65 Serine 9.15 5.68 Threonine 2.09 9.10 5.60 *In all cases p orresponds to ionization of the carboxyl group; paz corresponds to deprotonation of the ammonium ion TABLE 27.3 Acid-Base Properties of Amino Acids with ionizable Side Amino acid Aspartic acid 1.88 2.77 Glutamic acid 2.19 9.67 yrosine 2.20 2.18 1248 Histidyl 9.17 7.59 *In all cases pKa1 corresponds to ionization of the carboxyl group of RCHCO2H PROBLEM 27.3 Write the most stable structural formula for tyrosine (aIn its cationic form (c As a monoanion (b) In its zwitterionic form SAMPLE SOLUTION (a)The cationic form of tyrosine is the one present at low pH. The positive charge is on nitrogen, and the species present is an ammonium HO- CH2 CHCO2H Back Forward Main MenuToc Study Guide ToC Student o MHHE Website
PROBLEM 27.3 Write the most stable structural formula for tyrosine: (a) In its cationic form (c) As a monoanion (b) In its zwitterionic form (d) As a dianion SAMPLE SOLUTION (a) The cationic form of tyrosine is the one present at low pH. The positive charge is on nitrogen, and the species present is an ammonium ion. HO CH2CHCO2H NH3 27.3 Acid–Base Behavior of Amino Acids 1059 TABLE 27.2 Acid-Base Properties of Amino Acids with Neutral Side Chains Amino acid Glycine Alanine Valine Leucine Isoleucine Methionine Proline Phenylalanine Tryptophan Asparagine Glutamine Serine Threonine pKa1* 2.34 2.34 2.32 2.36 2.36 2.28 1.99 1.83 2.83 2.02 2.17 2.21 2.09 pKa2* 9.60 9.69 9.62 9.60 9.60 9.21 10.60 9.13 9.39 8.80 9.13 9.15 9.10 pI 5.97 6.00 5.96 5.98 6.02 5.74 6.30 5.48 5.89 5.41 5.65 5.68 5.60 *In all cases pKa1 corresponds to ionization of the carboxyl group; pKa2 corresponds to deprotonation of the ammonium ion. TABLE 27.3 Acid-Base Properties of Amino Acids with Ionizable Side Chains Amino acid Aspartic acid Glutamic acid Tyrosine Cysteine Lysine Arginine Histidine pKa1* 1.88 2.19 2.20 1.96 2.18 2.17 1.82 pKa2 3.65 4.25 9.11 8.18 8.95 9.04 6.00 pKa3 9.60 9.67 10.07 10.28 10.53 12.48 9.17 pI 2.77 3.22 5.66 5.07 9.74 10.76 7.59 *In all cases pKa1 corresponds to ionization of the carboxyl group of RCHCO2H. W NH3 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
1060 CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids ELECTROPHORESIS lectrophoresis is a method for separation and electrode. When the pH of the buffer corresponds to can charged particles in an electric field. Its principles migrate fromts acid has no net charge and does not purification that depends on the movement of the pl, the amin Thus if a mixture containing alani nine, aspartIc behavior of some representative amino acids. The acid, and lysine is subjected to electrophoresis in a medium is a cellulose acetate strip that is moistened buffer that matches the isoelectric point of alanine with an aqueous solution buffered at a particular pH. (pH 6.0), aspartic acid(pl 2 8)migrates toward the The opposite ends of the strip are placed in separate positive electrode, alanine remains at the origin, and compartments containing the buffer, and each com- lysine(pl =9.7) migrates toward the negative elec- partment is connected to a source of direct electric trode( Figure 27. 3b) current( Figure 27.3a). If the buffer solution is more acidic than the isoelectric point(pl) of the amino acid O2 CCH2 CHCO2 CH3 CHCO2 H3N(CH2)4CHCO2 he amino acid has a net positive charge and mi- grates toward the negatively charged electrode Con- NH3 +NH versely, when the buffer is more basic than the pl of Aspartic acid Alanine ysine he amino acid, the amino acid has a net negative (monoanion) (monocation) charge and migrates toward the positively charged Cont A mixture of amino acids O,CCHCHCO CHaCHCO H3N(CH,)CHCO? NH3 is placed at the center of a sheet of cellulose acetate. The sheet is soaked with an aqueous solution buffered at a pH of 6.0. At this pH aspartic acid O exists as its -I ion, alanine O as its zwitterion, and lysine O as its+I ion. Application of an electric current causes the negatively charged ions to migrate to the t electrode, and the ositively charged ions to migrate to the -electrode. The zwitterion, with a net charge of zero, remains at its original position. FIGURE 27.3 Application of electrophoresis to the separation of aspartic acid, alanine, and lysine according to their charge type at a pH corresponding to the isoelectric point (pn of alanine Back Forward Main MenuToc Study Guide ToC Student o MHHE Website
1060 CHAPTER TWENTY-SEVEN Amino Acids, Peptides, and Proteins. Nucleic Acids ELECTROPHORESIS Electrophoresis is a method for separation and purification that depends on the movement of charged particles in an electric field. Its principles can be introduced by considering the electrophoretic behavior of some representative amino acids. The medium is a cellulose acetate strip that is moistened with an aqueous solution buffered at a particular pH. The opposite ends of the strip are placed in separate compartments containing the buffer, and each compartment is connected to a source of direct electric current (Figure 27.3a). If the buffer solution is more acidic than the isoelectric point (pI) of the amino acid, the amino acid has a net positive charge and migrates toward the negatively charged electrode. Conversely, when the buffer is more basic than the pI of the amino acid, the amino acid has a net negative charge and migrates toward the positively charged electrode. When the pH of the buffer corresponds to the pI, the amino acid has no net charge and does not migrate from the origin. Thus if a mixture containing alanine, aspartic acid, and lysine is subjected to electrophoresis in a buffer that matches the isoelectric point of alanine (pH 6.0), aspartic acid (pI 2.8) migrates toward the positive electrode, alanine remains at the origin, and lysine (pI 9.7) migrates toward the negative electrode (Figure 27.3b). O2CCH2CHCO2 NH3 Aspartic acid (monoanion) CH3CHCO2 NH3 Alanine (neutral) H3N(CH2)4CHCO2 NH3 Lysine (monocation) A mixture of amino acids O2CCH2CHCO2 H3N(CH2)4CHCO2 CH3CHCO2 NH3 NH3 NH3 is placed at the center of a sheet of cellulose acetate. The sheet is soaked with an aqueous solution buffered at a pH of 6.0. At this pH aspartic acid exists as its 1 ion, alanine as its zwitterion, and lysine as its 1 ion. (a) (b) Application of an electric current causes the negatively charged ions to migrate to the electrode, and the positively charged ions to migrate to the electrode. The zwitterion, with a net charge of zero, remains at its original position. —Cont. FIGURE 27.3 Application of electrophoresis to the separation of aspartic acid, alanine, and lysine according to their charge type at a pH corresponding to the isoelectric point (pI) of alanine. Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website