chapter AMINO ACIDS, PEPTIDES AND PROTEINS 3.1 Amino Acids 75 acids, covalently linked in characteristic linear sequences 3.2 Peptides and Proteins 85 Because each of these amino acids has a side chain with 3.3 Working with Proteins 89 distinctive chemical properties, this group of 20 pre cursor molecules may be regarded as the alphabet in 3. 4 The covalent structure of proteins 96 which the language of protein structure is written 3.5 Protein Sequences and Evolution 10 What is most remarkable is that cells can produce proteins with strikingly different properties and activi- ties by joining the same 20 amino acids in many differ ent combinations and sequences From these building The word protein that I propose to you. . I would wish to blocks different organisms can make such widely diverse derive from proteios, because it appears to be the products as enzymes, hormones, antibodies, trans primitive or principal substance of animal nutrition that porters, muscle fibers, the lens protein of the eye, feat- plants prepare for the herbivores, and which the latter ers, spider webs, rhinoceros horn, milk proteins, antibi- then furnish to the carnivores otics, mushroom poisons, and myriad other substances biological activities(Fig 3-1). Among . Berzelius letter to G. J. Mulder. 1838 these protein products, the enzymes are the most var ied and specialized. Virtually all cellular reactions are oteins are the most abundant biological macromol- Protein structure and function are the topics of this ecules, occurring in all cells and all parts of cells. Pro- and the next three chapters. We begin with a descrip ins also occur in great variety; thousands of different tion of the fundamental chemical properties of amino kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in the millions, may be found in a single cell. Moreover, proteins exhibit enormous diversity of biological function and are the 3.1 Amino Acids ways discussed in Part Ill of this book. Proteins are the 0 Protein Architecture--Amino Acids molecular instruments through which genetic informa- Proteins are polymers of amino acids, with each amino tion is expressed acid residue joined to its neighbor by a specific type Relatively simple monomeric subunits provide the of covalent bond. (The term"residue"reflects the loss key to the structure of the thousands of different pro- of the elements of water when one amino acid is joined teins. All proteins, whether from the most ancient lines to another. Proteins can be broken down (hydrolyzed) of bacteria or from the most complex forms of life, are to their constituent amino acids by a variety of methods constructed from the same ubiquitous set of 20 amino and the earliest studies of proteins naturally focused on
chapter AMINO ACIDS, PEPTIDES, AND PROTEINS 3.1 Amino Acids 75 3.2 Peptides and Proteins 85 3.3 Working with Proteins 89 3.4 The Covalent Structure of Proteins 96 3.5 Protein Sequences and Evolution 106 The word protein that I propose to you . . . I would wish to derive from proteios, because it appears to be the primitive or principal substance of animal nutrition that plants prepare for the herbivores, and which the latter then furnish to the carnivores. —J. J. Berzelius, letter to G. J. Mulder, 1838 – + – + 3 75 Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of cells. Proteins also occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in the millions, may be found in a single cell. Moreover, proteins exhibit enormous diversity of biological function and are the most important final products of the information pathways discussed in Part III of this book. Proteins are the molecular instruments through which genetic information is expressed. Relatively simple monomeric subunits provide the key to the structure of the thousands of different proteins. All proteins, whether from the most ancient lines of bacteria or from the most complex forms of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in characteristic linear sequences. Because each of these amino acids has a side chain with distinctive chemical properties, this group of 20 precursor molecules may be regarded as the alphabet in which the language of protein structure is written. What is most remarkable is that cells can produce proteins with strikingly different properties and activities by joining the same 20 amino acids in many different combinations and sequences. From these building blocks different organisms can make such widely diverse products as enzymes, hormones, antibodies, transporters, muscle fibers, the lens protein of the eye, feathers, spider webs, rhinoceros horn, milk proteins, antibiotics, mushroom poisons, and myriad other substances having distinct biological activities (Fig. 3–1). Among these protein products, the enzymes are the most varied and specialized. Virtually all cellular reactions are catalyzed by enzymes. Protein structure and function are the topics of this and the next three chapters. We begin with a description of the fundamental chemical properties of amino acids, peptides, and proteins. 3.1 Amino Acids Protein Architecture—Amino Acids Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by a specific type of covalent bond. (The term “residue” reflects the loss of the elements of water when one amino acid is joined to another.) Proteins can be broken down (hydrolyzed) to their constituent amino acids by a variety of methods, and the earliest studies of proteins naturally focused on 8885d_c03_075 12/23/03 10:16 AM Page 75 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins FIGURE 3-1 Some functions of proteins. (a) The light prodt ers. The black rhinoceros is nearing extinction in the wild because of fireflies is the result of a reaction involving the protein luciferin and he belief prevalent in some parts of the world that a powder derived ATP, catalyzed by the enzyme luciferase(see Box 13-2).(b)Erythro- from its horn has aphrodisiac properties. In reality, the chemical prop- cytes contain large amounts of the oxy porting protein erties of powdered rhinoceros horn are no different from those of pow- moglobin. (c) The protein keratin, formed by all vertebrates, is the dered bovine hooves or human fingernails hief structural component of hair, scales, horn, wool, nails, and feath- the free amino acids derived from them. twenty differ- symbols (Table 3-1), which are used as shorthand to in- ent amino acids are commonly found in proteins. The dicate the composition and sequence of amino acids first to be discovered was asparagine, in 1806. The last polymerized in proteins of the 20 to be found, threonine, was not identified until Two conventions are used to identify the carbons in 1938. All the amino acids have trivial or common names, an amino acid-a practice that can be confusing. The in some cases derived from the source from which they additional carbons in an R group are commonly desig were first isolated. Asparagine was first found in as- nated B, 2,8, E, and so forth, proceeding out from the paragus, and glutamate in wheat gluten; tyrosine a carbon. For most other organic molecules, carbon first isolated from cheese (its name is derived from atoms are simply numbered from one end, giving high Greek tyros,"cheese ) and glycine ( Greek glykos, est priority(C-1)to the carbon with the substituent con- sweet) was so named because of its sweet taste taining the atom of highest atomic number. within this latter convention, the carboxyl carbon of an amino acid Amino Acids share Common structural Features would be c-1 and the a carbon would be c-2. in some all 20 of the common amino acids are a-amino acids cases, such as amino acids with heterocyclic r groups They have a carboxyl group and no group bonded the greek lettering system is ambiguous and the num- to the same carbon atom (the a carbon)(Fig. 3-2). They bering convention is therefore used differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge, and CH2-CH2-CH2-CH2-CH-CO0- which influence the solubility of the amino acids in wa- *NHs ter. In addition to these 20 amino acids there are many +NHs less common ones. Some are residues modified after a protein has been synthesized; others are amino acids For all the common amino acids except glycine, the present in living organisms but not as constituents of a carbon is bonded to four different groups: a carboxyl proteins. The common amino acids of proteins have group, an amino group, an R group, and a hydrogen atom been assigned three-letter abbreviations and one-letter (Fig. 3-2; in glycine, the R group is another hydrogen atom). The a-carbon atom is thus a chiral center (p. 17). Because of the tetrahedral arrangement of the bonding orbitals around the a-carbon atom, the four dif- ferent groups can occupy two unique spatial arrange- HaN-C-H stereoisomers. Since they are nonsuperimposable mir- or images of each other(Fig. 3-3), the two forms rep- FIGURE 3-2 General structure of an amino acid. This structure is resent a class of stereoisomers called enantiomers(see common to all but one of the a-amino acids (Proline, a cyclic amino Fig. 1-19). All molecules with a chiral center are also acid, is the exception. )The R group or side chain(red) attached to the optically active-that is, they rotate plane-polarized a carbon(blue) is different in each amino acid light(see Box 1-2)
the free amino acids derived from them. Twenty different amino acids are commonly found in proteins. The first to be discovered was asparagine, in 1806. The last of the 20 to be found, threonine, was not identified until 1938. All the amino acids have trivial or common names, in some cases derived from the source from which they were first isolated. Asparagine was first found in asparagus, and glutamate in wheat gluten; tyrosine was first isolated from cheese (its name is derived from the Greek tyros, “cheese”); and glycine (Greek glykos, “sweet”) was so named because of its sweet taste. Amino Acids Share Common Structural Features All 20 of the common amino acids are -amino acids. They have a carboxyl group and an amino group bonded to the same carbon atom (the carbon) (Fig. 3–2). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water. In addition to these 20 amino acids there are many less common ones. Some are residues modified after a protein has been synthesized; others are amino acids present in living organisms but not as constituents of proteins. The common amino acids of proteins have been assigned three-letter abbreviations and one-letter symbols (Table 3–1), which are used as shorthand to indicate the composition and sequence of amino acids polymerized in proteins. Two conventions are used to identify the carbons in an amino acid—a practice that can be confusing. The additional carbons in an R group are commonly designated , , , , and so forth, proceeding out from the carbon. For most other organic molecules, carbon atoms are simply numbered from one end, giving highest priority (C-1) to the carbon with the substituent containing the atom of highest atomic number. Within this latter convention, the carboxyl carbon of an amino acid would be C-1 and the carbon would be C-2. In some cases, such as amino acids with heterocyclic R groups, the Greek lettering system is ambiguous and the numbering convention is therefore used. For all the common amino acids except glycine, the carbon is bonded to four different groups: a carboxyl group, an amino group, an R group, and a hydrogen atom (Fig. 3–2; in glycine, the R group is another hydrogen atom). The -carbon atom is thus a chiral center (p. 17). Because of the tetrahedral arrangement of the bonding orbitals around the -carbon atom, the four different groups can occupy two unique spatial arrangements, and thus amino acids have two possible stereoisomers. Since they are nonsuperimposable mirror images of each other (Fig. 3–3), the two forms represent a class of stereoisomers called enantiomers (see Fig. 1–19). All molecules with a chiral center are also optically active—that is, they rotate plane-polarized light (see Box 1–2). CH2 NH3 COO NH3 CH2 CH2 CH2 CH Lysine 6 1 5 4 3 2 ed gba 76 Chapter 3 Amino Acids, Peptides, and Proteins (a) (b) (c) FIGURE 3–1 Some functions of proteins. (a) The light produced by fireflies is the result of a reaction involving the protein luciferin and ATP, catalyzed by the enzyme luciferase (see Box 13–2). (b) Erythrocytes contain large amounts of the oxygen-transporting protein hemoglobin. (c) The protein keratin, formed by all vertebrates, is the chief structural component of hair, scales, horn, wool, nails, and feathers. The black rhinoceros is nearing extinction in the wild because of the belief prevalent in some parts of the world that a powder derived from its horn has aphrodisiac properties. In reality, the chemical properties of powdered rhinoceros horn are no different from those of powdered bovine hooves or human fingernails. H3N C COO R H FIGURE 3–2 General structure of an amino acid. This structure is common to all but one of the -amino acids. (Proline, a cyclic amino acid, is the exception.) The R group or side chain (red) attached to the carbon (blue) is different in each amino acid. 8885d_c03_076 12/23/03 10:20 AM Page 76 mac111 mac111:reb:
3.1 Amino Acids Special nomenclature has been developed to spec CHO CHO ify the absolute configuration of the four substituents HO-C-H of asymmetric carbon atoms. The absolute configura- tions of simple sugars and amino acids are specified by OH CH2OH the D, L system(Fig. 3-4), based on the absolute con L-Glyceraldehyde acraldehyde figuration of the three-carbon sugar glyceraldehyde, a COr convention proposed by Emil Fischer in 1891.(Fischer H knew what groups surrounded the asymmetric carbon of glyceraldehyde but had to guess at their absolute H3 D-Alanine configuration; his guess was later confirmed by x-ray diffraction analysis. For all chiral compounds, stereo- FIGURE 3-4 Steric relationship of the stereoisomers of alanine to isomers having a configuration related to that of the absolute configuration of L-and D-glyceraldehyde. In these per. L-glyceraldehyde are designated L, and stereoisomers spective formulas, the carbons are lined up vertically, with the chiral related to D-glyceraldehyde are designated D. The func atom in the center. The carbons in these molecules are numbered be. tional groups of L-alanine are matched with those of L- ginning with the terminal aldehyde or carboxyl carbon (red), 1 to 3 glyceraldehyde by aligning those that can be intercon- from top to bottom as shown. When presented in this way, the R group verted by simple, one-step chemical reactions. Thus the of the amino acid (in this case the methyl group of alanine) is always carboxyl group of L-alanine occupies the same position below the a carbon L-Amino acids are those with the a-amino group about the chiral carbon as does the aldehyde group on the left, and D-amino acids have the a-amino group on the right of L-glyceraldehyde, because an aldehyde is readily converted to a carboxyl group via a one-step oxidation. Historically, the similar l and d designations were used L-amino acids are levorotatory, and the convention for levorotatory(rotating light to the left) and dextro- shown in Figure 3-4 was needed to avoid potential am rotatory(rotating light to the right). However, not all biguities about absolute configuration. By Fischer's con vention, L and D refer only to the absolute configura- tion of the four substituents around the chiral carbon COo not to optical properties of the molecule. Another system of specifying configuration around @H∈@→阻 a chiral center is the Rs system, which is used in the systematic nomenclature of organic chemistry and de- scribes more precisely the configuration of molecules CH with more than one chiral center(see p. 18) Alanine The Amino Acid residues in proteins Are L Stereoiso COo COo Nearly all biological compounds with a chiral center oc cur naturally in only one stereoisomeric form, either D or L. The amino acid residues in protein molecules are L-Alanine D-Alanine exclusively L stereoisomers. D-Amino acid residues have nly in COO cluding some peptides of bacterial cell walls and certain H3N-C-H -NH3 peptide antibiotics It is remarkable that virtually all amino acid residues H in proteins are L stereoisomers. When chiral compounds (c) D-Alanine are formed by ordinary chemical reactions, the result is racemic mixture of d and l isomers which are dififi- FIGURE 3-3 Stereoisomerism in a-amino acids. (a)The two stereoiso- cult for a chemist to distinguish and separate. but to a ages of each other (enantiomers).(b, c) Two different conventions for living system, D and L isomers are as different as the showing the configurations in space of stereoisomers In perspective right hand and the left. The formation of stable,re- formulas(b)the solid wedge-shaped bonds project out of the plane peating substructures in proteins(Chapter 4)generally of the paper, the dashed bonds behind it. In projection formulas(c) requires that their constituent amino acids be of one the horizontal bonds are assumed to project out of the plane of the stereochemical series. Cells are able to specifically syn- paper, the vertical bonds behind. However, projection formulas are thesize the l isomers of amino acids because the active ten used casually and are not always intended to portray a specific sites of enzymes are asymmetric, causing the reactions ey cat
Special nomenclature has been developed to specify the absolute configuration of the four substituents of asymmetric carbon atoms. The absolute configurations of simple sugars and amino acids are specified by the D, L system (Fig. 3–4), based on the absolute configuration of the three-carbon sugar glyceraldehyde, a convention proposed by Emil Fischer in 1891. (Fischer knew what groups surrounded the asymmetric carbon of glyceraldehyde but had to guess at their absolute configuration; his guess was later confirmed by x-ray diffraction analysis.) For all chiral compounds, stereoisomers having a configuration related to that of L-glyceraldehyde are designated L, and stereoisomers related to D-glyceraldehyde are designated D. The functional groups of L-alanine are matched with those of Lglyceraldehyde by aligning those that can be interconverted by simple, one-step chemical reactions. Thus the carboxyl group of L-alanine occupies the same position about the chiral carbon as does the aldehyde group of L-glyceraldehyde, because an aldehyde is readily converted to a carboxyl group via a one-step oxidation. Historically, the similar l and d designations were used for levorotatory (rotating light to the left) and dextrorotatory (rotating light to the right). However, not all L-amino acids are levorotatory, and the convention shown in Figure 3–4 was needed to avoid potential ambiguities about absolute configuration. By Fischer’s convention, L and D refer only to the absolute configuration of the four substituents around the chiral carbon, not to optical properties of the molecule. Another system of specifying configuration around a chiral center is the RS system, which is used in the systematic nomenclature of organic chemistry and describes more precisely the configuration of molecules with more than one chiral center (see p. 18). The Amino Acid Residues in Proteins Are L Stereoisomers Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L. The amino acid residues in protein molecules are exclusively L stereoisomers. D-Amino acid residues have been found only in a few, generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics. It is remarkable that virtually all amino acid residues in proteins are L stereoisomers. When chiral compounds are formed by ordinary chemical reactions, the result is a racemic mixture of D and L isomers, which are difficult for a chemist to distinguish and separate. But to a living system, D and L isomers are as different as the right hand and the left. The formation of stable, repeating substructures in proteins (Chapter 4) generally requires that their constituent amino acids be of one stereochemical series. Cells are able to specifically synthesize the L isomers of amino acids because the active sites of enzymes are asymmetric, causing the reactions they catalyze to be stereospecific. 3.1 Amino Acids 77 (a) COO H3N CH3 CH3 C H H C COO L-Alanine D-Alanine NH3 H3N C COO CH3 H H C COO CH3 N H3 (b) L-Alanine D-Alanine H3N COO CH3 H HC COO CH3 N H3 L-Alanine D-Alanine C (c) FIGURE 3–3 Stereoisomerism in -amino acids. (a)The two stereoisomers of alanine, L- and D-alanine, are nonsuperimposable mirror images of each other (enantiomers). (b, c) Two different conventions for showing the configurations in space of stereoisomers. In perspective formulas (b) the solid wedge-shaped bonds project out of the plane of the paper, the dashed bonds behind it. In projection formulas (c) the horizontal bonds are assumed to project out of the plane of the paper, the vertical bonds behind. However, projection formulas are often used casually and are not always intended to portray a specific stereochemical configuration. HO C 1 CHO 3 CH2OH H HC CHO CH2OH OH H3N C COO CH3 H HC COO CH3 N H3 L-Glyceraldehyde D-Alanine 2 D-Glyceraldehyde L-Alanine FIGURE 3–4 Steric relationship of the stereoisomers of alanine to the absolute configuration of L- and D-glyceraldehyde. In these perspective formulas, the carbons are lined up vertically, with the chiral atom in the center. The carbons in these molecules are numbered beginning with the terminal aldehyde or carboxyl carbon (red), 1 to 3 from top to bottom as shown. When presented in this way, the R group of the amino acid (in this case the methyl group of alanine) is always below the carbon. L-Amino acids are those with the -amino group on the left, and D-amino acids have the -amino group on the right. 8885d_c03_077 12/23/03 10:20 AM Page 77 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3-1 Properties and Conventions Associated with the Common Amino Acids Found in Prote ves Hydropathy Occurrence in Amino acid M, (-CO0H)(NH3)(R group) pl index* proteins(%)T Nonpolar, aliphatic Glycine 5.97 Alanine Ala A 2.34 9.69 6.01 1.8 7.8 P11519910 6.48 1.6 Valine 117 2.32 597 4.2 6.6 Leucine Leu l 131 2.36 9.60 Isoleucine Methionine Met M 149 2.28 9.21 5.74 1.9 2.3 Aromatic R groups Phenylalanine Phe F 651.83 9.13 5.48 2.8 lyrosine 181 Tryptophan 204 2.38 9.39 0.9 1.4 Polar, uncharged R groups Serine Ser s 9.15 5.68 0.8 Threonine Thr T 119 2.11 9.62 0.7 59 121 196 10.28 8.18 19 Asparagine Asn N 132 2.02 8.80 3.5 4.3 2.17 5.65 Positively charged R groups sine 462.18 8.95 10.53 9.74 3.9 59 Histidine 1.82 6.00 -3.2 Arg R 174 2.17 9.0412.48 10.76 5 5.1 Negatively charged R groups Asp D 133 188 3.65 2.77 3.5 Glutamate Glu E 147 2.19 9.67 4.25 3.22 -3.5 6.3 "A scale combining hydrophobicity and hydroph icity of R groups it can be used to measure the tendency of an amino acid to seek an aqueous emironment(- values)or a hydrophobic environment (t values). See Chapter 11. From Kyte, I Doolittle, RE(1982)A simple method for displaying the hydropathic character of a protein. J Mol Biol. 157, 105-132. Amino Acids Can Be Classified by R Group listed in Table 3-l. Within each class there are grada- Knowledge of the chemical properties of the common tions of polarity, size, and shape of the r groups amino acids is central to an understanding of biochem- Nonpolar, Aliphatic R Groups The r groups in this class of istry. The topic can be simplified by grouping the amino amino acids are nonpolar and hydrophobic. The side acids into five main classes based on the properties of chains of alanine, valine, leucine, and isoleucine their R groups (Table 3-1), in particular, their polarity, tend to cluster together within proteins, stabilizing pro- or tendency to interact with water at biological pH (near tein structure by means of hydrophobic interactions pH 7.0). The polarity of the R groups varies widely, from Glycine has the simplest structure. Although it is for- nonpolar and hydrophobic (water-insoluble) to highly mally nonpolar, its very small side chain makes no real polar and hydrophilic (water-soluble) contribution to hydrophobic interactions. Methionine, The structures of the 20 common amino acids are one of the two sulfur-containing amino acids, has a non- shown in Figure 3-5, and some of their properties are polar thioether group in its side chain. Proline has an
Amino Acids Can Be Classified by R Group Knowledge of the chemical properties of the common amino acids is central to an understanding of biochemistry. The topic can be simplified by grouping the amino acids into five main classes based on the properties of their R groups (Table 3–1), in particular, their polarity, or tendency to interact with water at biological pH (near pH 7.0). The polarity of the R groups varies widely, from nonpolar and hydrophobic (water-insoluble) to highly polar and hydrophilic (water-soluble). The structures of the 20 common amino acids are shown in Figure 3–5, and some of their properties are listed in Table 3–1. Within each class there are gradations of polarity, size, and shape of the R groups. Nonpolar, Aliphatic R Groups The R groups in this class of amino acids are nonpolar and hydrophobic. The side chains of alanine, valine, leucine, and isoleucine tend to cluster together within proteins, stabilizing protein structure by means of hydrophobic interactions. Glycine has the simplest structure. Although it is formally nonpolar, its very small side chain makes no real contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino acids, has a nonpolar thioether group in its side chain. Proline has an 78 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–1 Properties and Conventions Associated with the Common Amino Acids Found in Proteins pKa values Abbreviation/ pK1 pK2 pKR Hydropathy Occurrence in Amino acid symbol Mr (OCOOH) (ONH3 ) (R group) pI index* proteins (%)† Nonpolar, aliphatic R groups Glycine Gly G 75 2.34 9.60 5.97 0.4 7.2 Alanine Ala A 89 2.34 9.69 6.01 1.8 7.8 Proline Pro P 115 1.99 10.96 6.48 1.6 5.2 Valine Val V 117 2.32 9.62 5.97 4.2 6.6 Leucine Leu L 131 2.36 9.60 5.98 3.8 9.1 Isoleucine Ile I 131 2.36 9.68 6.02 4.5 5.3 Methionine Met M 149 2.28 9.21 5.74 1.9 2.3 Aromatic R groups Phenylalanine Phe F 165 1.83 9.13 5.48 2.8 3.9 Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 1.3 3.2 Tryptophan Trp W 204 2.38 9.39 5.89 0.9 1.4 Polar, uncharged R groups Serine Ser S 105 2.21 9.15 5.68 0.8 6.8 Threonine Thr T 119 2.11 9.62 5.87 0.7 5.9 Cysteine Cys C 121 1.96 10.28 8.18 5.07 2.5 1.9 Asparagine Asn N 132 2.02 8.80 5.41 3.5 4.3 Glutamine Gln Q 146 2.17 9.13 5.65 3.5 4.2 Positively charged R groups Lysine Lys K 146 2.18 8.95 10.53 9.74 3.9 5.9 Histidine His H 155 1.82 9.17 6.00 7.59 3.2 2.3 Arginine Arg R 174 2.17 9.04 12.48 10.76 4.5 5.1 Negatively charged R groups Aspartate Asp D 133 1.88 9.60 3.65 2.77 3.5 5.3 Glutamate Glu E 147 2.19 9.67 4.25 3.22 3.5 6.3 *A scale combining hydrophobicity and hydrophilicity of R groups; it can be used to measure the tendency of an amino acid to seek an aqueous environment ( values) or a hydrophobic environment ( values). See Chapter 11. From Kyte, J. & Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. † Average occurrence in more than 1,150 proteins. From Doolittle, R.F. (1989) Redundancies in protein sequences. In Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G.D., ed.), pp. 599–623, Plenum Press, New York. 8885d_c03_078 12/23/03 10:20 AM Page 78 mac111 mac111:reb:
3.1Ar Nonpolar, aliphatic R groups Aromatic r COO COO COO H小C-HH2NC Hs N-C-H H3N-C-H H3N-C--H HSN-C-H CHs CH CHO CH2 HC Glycine Alanine Proline COO CoO ISN- HSN-C-H Phenylalanine Tryptophan CH2 CH CHs CH positively charged R CHs COO Coo COO Leucin Isoleucine Methionine HoN-C-H HON-C-H CHa Polar, uncharged R groups CH2 COO COO H HoN-C-H H CH2 NH CH,O H-C-OHI CHo C=. CHs SHI NHo Serine Threonine L Histidine COO Coo Negatively charged R groups ISN- Coo CH2 CH2 H2N CH. Glutamine Aspartate FIGURE 3-5 The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH small but significant fraction 7.0. The unshaded portions are those common to all the amino acids; pH 7.0 the portions shaded in red are the R groups. Although the R group of aliphatic side chain with a distinctive cyclic structure. The tional group in some enzymes Tyrosine and tryptophan secondary amino (imino) group of proline residues is are significantly more polar than phenylalanine, because held in a rigid conformation that reduces the structural of the tyrosine hydroxyl group and the nitrogen of the flexibility of polypeptide regions containing proline tryptophan indole ring tryptophan and tyrosine, and to a much lesser ex Aromatic R Groups Phenylalanine, tyrosine, and tryp- tent phenylalanine, absorb ultraviolet light (Fig. 3-6 tophan, with their aromatic side chains, are relatively Box 3-1). This accounts for the characteristic strong ab- nonpolar (hydrophobic). All can participate in hy- sorbance of light by most proteins at a wavelength of drophobic interactions. The hydroxyl group of tyrosine 280 nm, a property exploited by researchers in the char- can form hydrogen bonds, and it is an important func acterization of proteins
aliphatic side chain with a distinctive cyclic structure. The secondary amino (imino) group of proline residues is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline. Aromatic R Groups Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are relatively nonpolar (hydrophobic). All can participate in hydrophobic interactions. The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important functional group in some enzymes. Tyrosine and tryptophan are significantly more polar than phenylalanine, because of the tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. Tryptophan and tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet light (Fig. 3–6; Box 3–1). This accounts for the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the characterization of proteins. 3.1 Amino Acids 79 Nonpolar, aliphatic R groups H3N C COO H H H3N C COO CH3 H H3N C COO C CH3 CH3 H H Glycine Alanine Valine Aromatic R groups H3N C COO CH2 H H3N C COO CH2 H OH Phenylalanine Tyrosine H2N H2C C COO H C CH2 H 2 Proline H3N C COO C C CH H2 H NH Tryptophan Polar, uncharged R groups H3N C COO CH2OH H H3N C COO H C CH3 OH H H3N C COO C SH H2 H Serine Threonine H3N C COO C C H2N O H2 H H3N C COO C C C H2N O H2 H2 H Positively charged R groups N C C C C H3N C COO H H2 H2 H2 H2 H3 C N C C C H3N C COO H H2 H2 H2 H NH2 N H2 H3N C COO C C NH H 2 H C H N Lysine Arginine Histidine Negatively charged R groups H3N C COO C COO H2 H H3N C COO C C COO H2 H2 H Asparagine Glutamine Aspartate Glutamate Cysteine CH H3N C COO C C CH3 CH3 H H2 H Leucine H3N C COO C C S CH3 H2 H2 H Methionine H3 C COO H C C CH3 H2 H H Isoleucine N C 3 FIGURE 3–5 The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in red are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3–1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. 8885d_c03_079 12/23/03 10:20 AM Page 79 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins Polar, Uncharged R Groups The r groups of these amino COO CO0- acids are more soluble in water, or more hydrophilic H3N-CH H3N一CH than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water This class of amino acids includes serine CH22H**+2e SH threonine, cysteine, asparagine, and glutamine. The polarity of serine and threonine is contributed by 2H++2e their hydroxyl groups: that of cysteine by its sulfhydryl CH group; and that of asparagine and glutamine by their Cyst amide groups. CH一NH3 Asparagine and glutamine are the amides of two COO CO0- other amino acids also found in proteins, aspartate and FIGURE 3-7 Reversible formation of a disulfide bond by the oxida glutamate, respectively, to which asparagine and gluta- tion of two molecules of cysteine. Disulfide bonds between Cys mine are easily hydrolyzed by acid or base. Cysteine Is residues stabilize the structures of many proteins readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine mole cules or residues are joined by a disulfide bond(Fig 3-7). The disulfide-linked residues are strongly hy- tion on its aliphatic chain; nine, which has a pos drophobic(nonpolar) Disulfide bonds play a special tively charged guanidino and histidine, which ole in the structures of many proteins by forming co- has an imidazole group is the only common valent links between parts of a protein molecule or be- amino acid having an ionizable side chain with a pk tween two different polypeptide chains near neutrality. In many enzyme-catalyzed reactions, a Positively Charged( Basic)R Groups The most hydrophilic His residue facilitates the reaction by serving as a pro- ton donor/acceptor. R groups are those that are either positively tively charged. The amino acids in which the r groups Negatively charged (Acidic)R Groups The two amind have significant positive charge at pH 7.0 are lysine, having R groups with a net negative charge at pH 7.0 which has a second primary amino group at the g posi- are aspartate and glutamate, each of which has a sec- ond carboxyl group Uncommon Amino Acids Also have Important Functions In addition to the 20 common amino acids, proteins may contain residues created by modification of com- mon residues already incorporated into a polypeptide (Fig. 3-8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-N Methyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is y-carboxyglutamate, found in the blood- clotting protein prothrombin and in certain other pro- teins that bind Ca+ as part of their biological function 230240250260270280290300310 More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids. Selenocysteine is a special case. This rare amino Comparison of the light absorption spectra of the aromatic amino acids acid residue is introduced during protein synthesis tryptophan and tyrosine at pH 6.0. The amino acids are present in rather than created through a postsynthetic modifica- equimolar amounts(10-3M) under identical conditions. The meas- tion. It contains selenium rather than the sulfur of cys- ed absorbance of tryptophan is as much as four times that of tyro. teine. Actually derived from serine, selenocysteine is a sine. Note that the maximum light absorption for both tryptophan and constituent of just a few known proteins tyrosine occurs near a wavelength of 280 nm. Light absorption by the Some 300 additional amino acids have been found third aromatic amino acid, phenylalanine (not shown), generally con. in cells. They have a variety of functions but are not tributes little to the spectroscopic properties of proteins. constituents of proteins. Ornithine and citrulline
Polar, Uncharged R Groups The R groups of these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water. This class of amino acids includes serine, threonine, cysteine, asparagine, and glutamine. The polarity of serine and threonine is contributed by their hydroxyl groups; that of cysteine by its sulfhydryl group; and that of asparagine and glutamine by their amide groups. Asparagine and glutamine are the amides of two other amino acids also found in proteins, aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules or residues are joined by a disulfide bond (Fig. 3–7). The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains. Positively Charged (Basic) R Groups The most hydrophilic R groups are those that are either positively or negatively charged. The amino acids in which the R groups have significant positive charge at pH 7.0 are lysine, which has a second primary amino group at the position on its aliphatic chain; arginine, which has a positively charged guanidino group; and histidine, which has an imidazole group. Histidine is the only common amino acid having an ionizable side chain with a pKa near neutrality. In many enzyme-catalyzed reactions, a His residue facilitates the reaction by serving as a proton donor/acceptor. Negatively Charged (Acidic) R Groups The two amino acids having R groups with a net negative charge at pH 7.0 are aspartate and glutamate, each of which has a second carboxyl group. Uncommon Amino Acids Also Have Important Functions In addition to the 20 common amino acids, proteins may contain residues created by modification of common residues already incorporated into a polypeptide (Fig. 3–8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-NMethyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is -carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2 as part of their biological function. More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. Selenocysteine is a special case. This rare amino acid residue is introduced during protein synthesis rather than created through a postsynthetic modification. It contains selenium rather than the sulfur of cysteine. Actually derived from serine, selenocysteine is a constituent of just a few known proteins. Some 300 additional amino acids have been found in cells. They have a variety of functions but are not constituents of proteins. Ornithine and citrulline 80 Chapter 3 Amino Acids, Peptides, and Proteins Tryptophan Wavelength (nm) Absorbance 5 4 3 2 1 0 6 230 240 250 260 270 280 290 300 310 Tyrosine FIGURE 3–6 Absorption of ultraviolet light by aromatic amino acids. Comparison of the light absorption spectra of the aromatic amino acids tryptophan and tyrosine at pH 6.0. The amino acids are present in equimolar amounts (103 M) under identical conditions. The measured absorbance of tryptophan is as much as four times that of tyrosine. Note that the maximum light absorption for both tryptophan and tyrosine occurs near a wavelength of 280 nm. Light absorption by the third aromatic amino acid, phenylalanine (not shown), generally contributes little to the spectroscopic properties of proteins. CH 2H 2e 2H 2e COO COO H3N CH2 CH CH2 SH SH Cysteine Cystine Cysteine NH3 CH COO COO H3N CH2 CH CH2 S S NH3 FIGURE 3–7 Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins. 8885d_c03_080 12/23/03 10:20 AM Page 80 mac111 mac111:reb:
3.1Ar FIGURE 3-8 Uncommon amino acids. (a)Some uncommon amino CH2 acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in CH-COO red. Desmosine is formed from four Lys residues(the four carbon back bones are shaded in yellow). Note the use of either numbers or Greek ify the carbon atoms in these structures. (b)Ornithin 4-Hydroxyproline and citrulline, which are not found in proteins, are intermediates in the biosynthesis of arginine and in the urea cycle. H3N一CH2CH—CH2-CH2-CH-COO Hs 5-Hydroxylysin CHs-NH-CH2-CH2-CH2-CH2--CH--Co0 HoN-C-H HaN-C--H 6-N-Methyllysine R Nonionic Zwitterionic form form COO OOC-CH-CHo-CH-COo FIGURE 3-9 Nonionic and zwitterionic forms of amino acids. The nonionic form does not significant amounts in aqueous so NHs utions. The zwitterion predominates at neutral pl y-Carboxyglutamate Coo Amino Acids Can Act as Acids and bases When an amino acid is dissolved in water, it exists in so- HBN (CH2)3 NHs lution as the dipolar ion, or zwitterion (German for (CH2)2-CH hybrid ion"), shown in Figure 3-9. A zwitterion can act CO0 as either an acid (proton donor) (CH2)4 R--C-Co0-=R-C-Co0-+H+ NH Desmosine H Zwitterion HSe-CH,-CH-cOo or a base(proton acceptor): NH NHs NHs H.N-CHo-CHo-CHo-CH Ornithine Substances having this dual nature are amphoteric and are often called ampholytes (from"amphoteric lectrolytes"). A simple monoamino monocarboxylic H2N-C-N-CH2--CH2-CH2-CH-Co0 amino acid, such as alanine, is a diprotic acid when fully protonated--it has two groups, the-COoH group and Citrulline the -NH3 group, that can yield protons (Fig. 3-8b) deserve special note because they are key R--C-COOH intermediates(metabolites) in the biosynthesis of argi- Net NHs nine(Chapter 22) and in the urea cycle(Chapter 18). charge:+1
Amino Acids Can Act as Acids and Bases When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion (German for “hybrid ion”), shown in Figure 3–9. A zwitterion can act as either an acid (proton donor): or a base (proton acceptor): Substances having this dual nature are amphoteric and are often called ampholytes (from “amphoteric electrolytes”). A simple monoamino monocarboxylic - amino acid, such as alanine, is a diprotic acid when fully protonated—it has two groups, the OCOOH group and the ONH3 group, that can yield protons: H R C COOH H R C COO NH3 H NH3 Zwitterion H R C COO NH3 H R C COO NH2 H Zwitterion 3.1 Amino Acids 81 H3N CH2 CH2 CH2 C NH3 H COO Ornithine H2N C O N H CH2 CH2 CH2 C NH3 H COO (b) Citrulline HO C H H2C N H H C CH2 H COO 4-Hydroxyproline H3N CH2 C OH H CH2 CH2 C NH3 H COO 5-Hydroxylysine CH3 NH CH2 CH2 CH2 CH2 CH COO 6-N-Methyllysine OOC C COO H CH2 C NH3 H COO -Carboxyglutamate C H3N OOC H (CH2)2 C H3N COO H (CH2)3 C N H3 COO H C (C N H2)4 H3N COO H Desmosine HSe CH2 C NH3 H COO (a) Selenocysteine (CH2)2 NH3 FIGURE 3–8 Uncommon amino acids. (a) Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the four carbon backbones are shaded in yellow). Note the use of either numbers or Greek letters to identify the carbon atoms in these structures. (b) Ornithine and citrulline, which are not found in proteins, are intermediates in the biosynthesis of arginine and in the urea cycle. H R C COO H R C COOH NH3 NH3 1 0 1 H H R C COO NH2 H Net charge: H2N C C R H H3N C C R H Nonionic form Zwitterionic form O HO O O FIGURE 3–9 Nonionic and zwitterionic forms of amino acids. The nonionic form does not occur in significant amounts in aqueous solutions. The zwitterion predominates at neutral pH. (Fig. 3–8b) deserve special note because they are key intermediates (metabolites) in the biosynthesis of arginine (Chapter 22) and in the urea cycle (Chapter 18). 8885d_c03_081 12/23/03 10:21 AM Page 81 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins BOX 3-1 WORKING IN BIOCHEMISTRY Absorption of Light by Molecules moles per liter), and l is the path length of the light The Lambert-Beer Law absorbing sample (in centimeters). The Lambert-Beer A wide range of biomolecules absorb light at charac- monochromatic (of a single wavelength) and that the law assumes that the incident light is parallel and teristic wavelengths, just as tryptophan absorbs light 280 nm(see Fig. 3-6). Measurement of light absorp- solvent and solute molecules are randomly oriented. tion by a spectrophotometer is used to detect and iden- The expression log(o/I)is called the absorbance tify molecules and to measure their concentration in designated A It is important to note that each successive milli- solution. The fraction of the incident light absorbed by meter of path length of absorbing solution in a 1.0 cm a solution at a given wavelength is related to the thick ness of the absorbing layer(path length) and the con- cell absorbs not a constant amount but a constant frac tion of the light that is incident upon it. However, with centration of the absorbing species(Fig. 1). These two an absorbing layer of fixed path length, the ab- relationships are combined into the Lambert-Beer \n SOrbance, A, is directly proportional to the con- centration of the absorbing solute. The molar extinction coefficient varies with the where lo is the intensity of the incident light, I is the in- nature of the absorbing compound, the solvent, and tensity of the transmitted light, a is the molar extinc- the wavelength, and also with pH if the light-absorbing tion coefficient (in units of liters per mole-centimeter), species is in equilibrium with an ionization state that c is the concentration of the absorbing species (in has different absorbance properties URE 1 The principal components of a ntensity of spectrophotometer. A light source emits light along a broad spectrum, then the monochromator selects and transmits light of a particular wavelength. The monochro- mA=0.0 matic light passes through the sample in a cuvette of path length / and is absorbed by Lamp Monochromator the sample in proportion to the concentra- Sample cuvette tion of the absorbing species. The transmit rth c moles/liter ted light is measured by a detector. of absorbing species Amino Acids have characteristic Titration curves (Recall from Chapter 2 that pH and pka are simply con venient notations for proton concentration and the Acid-base titration involves the gradual addition or re- equilibrium constant for ionization, respectively. The noval of protons(Chapter 2). Figure 3-10 shows the pKa is a measure of the tendency of a group to give up titration curve of the diprotic form of glycine. The plot a proton, with that tendency decreasing tenfold as the has two distinct stages, corresponding to deprotonation pKa increases by one unit. As the titration proceeds of two different groups on glycine. Each of the two another important point is reached at pH 5.97.Here stages resembles in shape the titration curve of a there is another point of inflection, at which removal of monoprotic acid, such as acetic acid (see Fig 2-10, the first proton is essentially complete and removal of and can be analyzed in the same way. At very low pH, the second has just begun. At this ph glycine is the predominant ionic species of glycine is the fully pro- present largely as the dipolar ionTH3N--CH2-CO0 tonated form, H3N--CH2--COOH. At the midpoint in We shall return to the significance of this inflection the first stage of the titration, in which the -CooH point in the titration curve (labeled pl in Fig. 3-10) group of glycine loses its proton, equimolar concentra- shortly. tions of the proton-donor (H3N--CH2--COOH) and The second stage of the titration corresponds to the proton-acceptor (H3N--CH2-CO0) species are removal of a proton from the -NH3 group of glycine present At the midpoint of any titration, a point of in- The ph at the midpoint of this stage is 9.60, equal to flection is reached where the pH is equal to the pka of the pka (labeled pk, in Fig. 3-10) for the--NH3 group the protonated group being titrated(see Fig 2-18). For The titration is essentially complete at a pH of about 12 glycine, the ph at the midpoint is 2.34, thus its-COoH at which point the predominant form of glycine is group has a pKa (labeled pKi in Fig. 3-10) of 2.34. H2N-CH2--CO0
Amino Acids Have Characteristic Titration Curves Acid-base titration involves the gradual addition or removal of protons (Chapter 2). Figure 3–10 shows the titration curve of the diprotic form of glycine. The plot has two distinct stages, corresponding to deprotonation of two different groups on glycine. Each of the two stages resembles in shape the titration curve of a monoprotic acid, such as acetic acid (see Fig. 2–17), and can be analyzed in the same way. At very low pH, the predominant ionic species of glycine is the fully protonated form, H3NOCH2 OCOOH. At the midpoint in the first stage of the titration, in which the OCOOH group of glycine loses its proton, equimolar concentrations of the proton-donor (H3NOCH2OCOOH) and proton-acceptor (H3NOCH2OCOO) species are present. At the midpoint of any titration, a point of inflection is reached where the pH is equal to the pKa of the protonated group being titrated (see Fig. 2–18). For glycine, the pH at the midpoint is 2.34, thus its OCOOH group has a pKa (labeled pK1 in Fig. 3–10) of 2.34. (Recall from Chapter 2 that pH and pKa are simply convenient notations for proton concentration and the equilibrium constant for ionization, respectively. The pKa is a measure of the tendency of a group to give up a proton, with that tendency decreasing tenfold as the pKa increases by one unit.) As the titration proceeds, another important point is reached at pH 5.97. Here there is another point of inflection, at which removal of the first proton is essentially complete and removal of the second has just begun. At this pH glycine is present largely as the dipolar ion H3NOCH2OCOO. We shall return to the significance of this inflection point in the titration curve (labeled pI in Fig. 3–10) shortly. The second stage of the titration corresponds to the removal of a proton from the ONH3 group of glycine. The pH at the midpoint of this stage is 9.60, equal to the pKa (labeled pK2 in Fig. 3–10) for the ONH3 group. The titration is essentially complete at a pH of about 12, at which point the predominant form of glycine is H2NOCH2OCOO. 82 Chapter 3 Amino Acids, Peptides, and Proteins BOX 3–1 WORKING IN BIOCHEMISTRY Absorption of Light by Molecules: The Lambert-Beer Law A wide range of biomolecules absorb light at characteristic wavelengths, just as tryptophan absorbs light at 280 nm (see Fig. 3–6). Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution. The fraction of the incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species (Fig. 1). These two relationships are combined into the Lambert-Beer law, log cl where I0 is the intensity of the incident light, I is the intensity of the transmitted light, is the molar extinction coefficient (in units of liters per mole-centimeter), c is the concentration of the absorbing species (in moles per liter), and l is the path length of the lightabsorbing sample (in centimeters). The Lambert-Beer law assumes that the incident light is parallel and monochromatic (of a single wavelength) and that the solvent and solute molecules are randomly oriented. The expression log (I0 /I) is called the absorbance, designated A. It is important to note that each successive millimeter of path length of absorbing solution in a 1.0 cm cell absorbs not a constant amount but a constant fraction of the light that is incident upon it. However, with an absorbing layer of fixed path length, the absorbance, A, is directly proportional to the concentration of the absorbing solute. The molar extinction coefficient varies with the nature of the absorbing compound, the solvent, and the wavelength, and also with pH if the light-absorbing species is in equilibrium with an ionization state that has different absorbance properties. I0 I Intensity of transmitted light I Lamp Monochromator Detector Intensity of incident light I0 Sample cuvette with c moles/liter of absorbing species A = 0.012 l FIGURE 1 The principal components of a spectrophotometer. A light source emits light along a broad spectrum, then the monochromator selects and transmits light of a particular wavelength. The monochromatic light passes through the sample in a cuvette of path length l and is absorbed by the sample in proportion to the concentration of the absorbing species. The transmitted light is measured by a detector. 8885d_c03_082 12/23/03 10:21 AM Page 82 mac111 mac111:reb:
3.1 Amino Acids N From the titration curve of glycine we can derive several important pieces of information. First, it gives a CH2 quantitative measure of the pka of each of the two ion- COOH COo COO izing groups: 2.34 for the-COOH group and 9.60 for the -NH group. Note that the carboxyl group of glycine is over 100 times more acidic(more easily ion 2=9.60 ized) than the carboxyl group of acetic acid, which, as we saw in Chapter 2, has a pKa of 4.76--about average for a carboxyl group attached to an otherwise unsub- stituted aliphatic hydrocarbon. The perturbed pKa of glycine is caused by repulsion between the departing proton and the nearby positively charged amino group on the a-carbon atom, as described in Figure 3-ll.The opposite charges on the resulting zwitterion are stabi lizing, nudging the equilibrium farther to the right. Sim- ilarly, the pka of the amino group in glycine is perturbed downward relative to the average pka of an amino group This effect is due partly to the electronegative oxygen atoms in the carboxyl groups, which tend to pull elec- trons toward them, increasing the tendency of the amino group to give up a proton. Hence, the a-amino group has a pKa that is lower than that of an aliphatic amine OH-(equivalents) such as methylamine (Fig. 3-11). In short, the pKa of FIGURE 3-10 Titration of an amino acid. Shown here is the titration any functional group is greatly affected by its chemical environment, a phenomenon sometimes exploited in the key points in the titration are shown above the graph. The shaded active sites of enzymes to promote exquisitely adapted boxes, centered at about pk,= 2.34 and pK2=9.60, indicate the re reaction mechanisms that depend on the perturbed pk gions of greatest buffering power. values of proton donor/acceptor groups of specific 8 Methyl-subs CH3--COoH CH.-CO0 CHa-NHs CHS--NH2 Acetic acid Methylamine The normal pka for a The normal pk, for a rboxyl group is about 4.8. mino group is about 10.6. Carboxyl and amino groups in glycine H-C--COOH H-C-COO H-C--CO0 H H H a-Amino acid(glycine) Electr in the carboxyl group pull electron lowers the pk for the carbo away from the amino group. and oppositely charged groups lower the pka by stabi FIGURE 3-11 Effect of the chemical environment on pK. The pKa perturbations of pKa are due to intramolecular interactions. Similar ef- values for the ionizable groups in glycine are lower than those for sim- fects can be caused by chemical groups that happen to be positioned ple, methyl-substituted amino and carboxyl groups. These downward earby-for example, in the active site of an enzyme
From the titration curve of glycine we can derive several important pieces of information. First, it gives a quantitative measure of the pKa of each of the two ionizing groups: 2.34 for the OCOOH group and 9.60 for the ONH3 group. Note that the carboxyl group of glycine is over 100 times more acidic (more easily ionized) than the carboxyl group of acetic acid, which, as we saw in Chapter 2, has a pKa of 4.76—about average for a carboxyl group attached to an otherwise unsubstituted aliphatic hydrocarbon. The perturbed pKa of glycine is caused by repulsion between the departing proton and the nearby positively charged amino group on the -carbon atom, as described in Figure 3–11. The opposite charges on the resulting zwitterion are stabilizing, nudging the equilibrium farther to the right. Similarly, the pKa of the amino group in glycine is perturbed downward relative to the average pKa of an amino group. This effect is due partly to the electronegative oxygen atoms in the carboxyl groups, which tend to pull electrons toward them, increasing the tendency of the amino group to give up a proton. Hence, the -amino group has a pKa that is lower than that of an aliphatic amine such as methylamine (Fig. 3–11). In short, the pKa of any functional group is greatly affected by its chemical environment, a phenomenon sometimes exploited in the active sites of enzymes to promote exquisitely adapted reaction mechanisms that depend on the perturbed pKa values of proton donor/acceptor groups of specific residues. 3.1 Amino Acids 83 N N C COOH H2 H3 C COO H2 H3 N C COO H2 H2 13 0.5 OH (equivalents) pH pI 5.97 0 0 7 1 1.5 2 pK1 pK2 pK2 9.60 pK1 2.34 Glycine FIGURE 3–10 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25 C. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about pK1 2.34 and pK2 9.60, indicate the regions of greatest buffering power. NH3 Methyl-substituted carboxyl and amino groups Acetic acid The normal pKa for a carboxyl group is about 4.8. pKa 2 4 6 8 10 12 Methylamine The normal pKa for an amino group is about 10.6. Carboxyl and amino groups in glycine -Amino acid (glycine) pKa 2.34 Repulsion between the amino group and the departing proton lowers the pKa for the carboxyl group, and oppositely charged groups lower the pKa by stabilizing the zwitterion. -Amino acid (glycine) pKa 9.60 Electronegative oxygen atoms in the carboxyl group pull electrons away from the amino group, lowering its pKa. CH3 COOH CH3 COO CH3 H C COO H NH2 H C COO H CH3 NH3 NH2 H H H C COOH H NH3 H H H H H H FIGURE 3–11 Effect of the chemical environment on pKa. The pKa values for the ionizable groups in glycine are lower than those for simple, methyl-substituted amino and carboxyl groups. These downward perturbations of pKa are due to intramolecular interactions. Similar effects can be caused by chemical groups that happen to be positioned nearby—for example, in the active site of an enzyme. 8885d_c03_083 12/23/03 10:21 AM Page 83 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins The second piece of information provided by the group in the range of 1.8 to 2.4, and pKa of the -NH3 titration curve of glycine is that this amino acid has two group in the range of 8.8 to 11.0(Table 3-1) egions of buffering power. One of these is the relatively Second, amino acids with an ionizable R group have flat portion of the curve, extending for approximately more complex titration curves, with three stages corre- 1 pH unit on either side of the first pKa of 2.34, indi- sponding to the three possible ionization steps; thus cating that glycine is a good buffer near this pH. The they have three pka values. The additional stage for the other buffering zone is centered around pH 9. 60. (Note titration of the ionizable r group merges to some extent that glycine is not a good buffer at the ph of intracel- with the other two. The titration curves for two amino lular fluid or blood, about 7.4.) Within the buffering acids of this type, glutamate and histidine, are shown in ranges of glycine, the Henderson-Hasselbalch equation Figure 3-12. The isoelectric points reflect the nature of (see Box 2-3) can be used to calculate the proportions the ionizing R groups present. For example, glutamate of proton-donor and proton-acceptor species of glycine equired to make a buffer at a given pH Titration Curves Predict the Electric Charge HSN-CH HIN-CH H,N-CH of Amino acids CH Another important piece of information derived from the titration curve of an amino acid is the relationship between its net electric charge and the ph of the solu- tion. At pH 5.97, the point of inflection between the 10 Glutamate two stages in its titration curve, glycine is present pre- dominantly as its dipolar form, fully ionized but with no 8 net electric charge(Fig. 3-10). The characteristic pH at which the net electric charge is zero is called the 6 isoelectric point or isoelectrie pH, designated pI For glycine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the two pka values p=(pK1+pk2)=b(234+9.60)=5.97 As is evident in Figure 3-10, glycine has a net negative charge at any pH above its pI and will thus move toward (a) the positive electrode(the anode) when placed in an electric field. At any pH below its pl, glycine has a net positive charge and will move toward the negative elec- trode (the cathode). The farther the pH of a glycine so- lution is from its isoelectric point, the greater the net electric charge of the population of glycine molecules At pH 1.0, for example, glycine exists almost entirely as 10 Histidine the form H3N--CH2-COOH, with a net positive charge of 1.0. At pH 2.34, where there is an equal mix ture of fHgN--CHo--COOH and fH3N-CH--CO0 the average or net positive charge is 0. 5. The sign and the magnitude of the net charge of any amino acid at any pH can be predicted in the same way. Amino Acids Differ in Their Acid-Base Properties The shared properties of many amino acids permit some simplifying generalizations about their acid-base behav iors. First, all amino acids with a single a-amino group a single a-carboxyl group, and an r group that does not OH(equivalents) ionize have titration curves resembling that of glycine (Fig. 3-10). These amino acids have very similar, al FIGURE 3-12 Titration curves for(a) glutamate and(b)histidine. The though not identical, pKa values: pKa of the-COOH pK, of the r group is designated here as pKg
group in the range of 1.8 to 2.4, and pKa of the ONH3 group in the range of 8.8 to 11.0 (Table 3–1). Second, amino acids with an ionizable R group have more complex titration curves, with three stages corresponding to the three possible ionization steps; thus they have three pKa values. The additional stage for the titration of the ionizable R group merges to some extent with the other two. The titration curves for two amino acids of this type, glutamate and histidine, are shown in Figure 3–12. The isoelectric points reflect the nature of the ionizing R groups present. For example, glutamate 84 Chapter 3 Amino Acids, Peptides, and Proteins 10 8 6 4 2 0 Glutamate H3N N N C COOH C C COOH H2 H2 H pK1 H3 C COO C C COOH H2 H2 H pKR H3 C COO C C COO H2 H2 H pK2 H2N C COO C C COO H2 H2 H pK2 9.67 pKR 4.25 pK1 2.19 1.0 2.0 3.0 pH OH (equivalents) (a) FIGURE 3–12 Titration curves for (a) glutamate and (b) histidine. The pKa of the R group is designated here as pKR. The second piece of information provided by the titration curve of glycine is that this amino acid has two regions of buffering power. One of these is the relatively flat portion of the curve, extending for approximately 1 pH unit on either side of the first pKa of 2.34, indicating that glycine is a good buffer near this pH. The other buffering zone is centered around pH 9.60. (Note that glycine is not a good buffer at the pH of intracellular fluid or blood, about 7.4.) Within the buffering ranges of glycine, the Henderson-Hasselbalch equation (see Box 2–3) can be used to calculate the proportions of proton-donor and proton-acceptor species of glycine required to make a buffer at a given pH. Titration Curves Predict the Electric Charge of Amino Acids Another important piece of information derived from the titration curve of an amino acid is the relationship between its net electric charge and the pH of the solution. At pH 5.97, the point of inflection between the two stages in its titration curve, glycine is present predominantly as its dipolar form, fully ionized but with no net electric charge (Fig. 3–10). The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI. For glycine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the two pKa values: pI 1 2 (pK1 pK2) 1 2 (2.34 9.60) 5.97 As is evident in Figure 3–10, glycine has a net negative charge at any pH above its pI and will thus move toward the positive electrode (the anode) when placed in an electric field. At any pH below its pI, glycine has a net positive charge and will move toward the negative electrode (the cathode). The farther the pH of a glycine solution is from its isoelectric point, the greater the net electric charge of the population of glycine molecules. At pH 1.0, for example, glycine exists almost entirely as the form H3NOCH2OCOOH, with a net positive charge of 1.0. At pH 2.34, where there is an equal mixture of H3NOCH2OCOOH and H3NOCH2OCOO, the average or net positive charge is 0.5. The sign and the magnitude of the net charge of any amino acid at any pH can be predicted in the same way. Amino Acids Differ in Their Acid-Base Properties The shared properties of many amino acids permit some simplifying generalizations about their acid-base behaviors. First, all amino acids with a single -amino group, a single -carboxyl group, and an R group that does not ionize have titration curves resembling that of glycine (Fig. 3–10). These amino acids have very similar, although not identical, pKa values: pKa of the OCOOH C H3N C COOH C CH C H N H2 H H3N C COO CH2 H H3N C COO CH2 H H2N C CH2 H pK1 1.82 pKR 6.0 pK2 9.17 C H N CH C H N H C H N CH C H N H C H N CH C H N 10 8 6 4 2 0 1.0 2.0 3.0 pH OH (equivalents) (b) COO H N Histidine pK1 pKR pK2 8885d_c03_084 12/23/03 10:21 AM Page 84 mac111 mac111:reb: