chap03 Amino acids, peptides and proteins

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 macromol-ecules, occurring in all cells and all parts of cells. Pro- teins 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 path- ways discussed in Part III of this book. Proteins are the molecular instruments through which genetic informa- tion is expressed. Relatively simple monomeric subunits provide the key to the structure of the thousands of different pro- teins. 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 pre- cursor 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 activi- ties by joining the same 20 amino acids in many differ- ent combinations and sequences. From these building blocks different organisms can make such widely diverse products as enzymes, hormones, antibodies, trans- porters, muscle fibers, the lens protein of the eye, feath- ers, spider webs, rhinoceros horn, milk proteins, antibi- otics, mushroom poisons, and myriad other substances having distinct biological activities (Fig. 3–1). Among these protein products, the enzymes are the most var- ied 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 descrip- tion 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: the free amino acids derived from them. Twenty differ- ent 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 as- paragus, 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 wa- ter. 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 in- dicate 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 desig- nated �, �, �, �, and so forth, proceeding out from the � carbon. For most other organic molecules, carbon atoms are simply numbered from one end, giving high- est priority (C-1) to the carbon with the substituent con- taining 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 num- bering 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 dif- ferent groups can occupy two unique spatial arrange- ments, and thus amino acids have two possible stereoisomers. Since they are nonsuperimposable mir- ror images of each other (Fig. 3–3), the two forms rep- resent 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 23456 1 e d g b a Chapter 3 Amino Acids, Peptides, and Proteins76 (a) (c)(b) 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) Erythro- cytes contain large amounts of the oxygen-transporting protein he- moglobin. (c) The protein keratin, formed by all vertebrates, is the chief structural component of hair, scales, horn, wool, nails, and feath- ers. 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 prop- erties of powdered rhinoceros horn are no different from those of pow- dered 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: Special nomenclature has been developed to spec- ify the absolute configuration of the four substituents of asymmetric carbon atoms. The absolute configura- tions of simple sugars and amino acids are specified by the D, L system (Fig. 3–4), based on the absolute con- figuration 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, stereo- isomers having a configuration related to that of L-glyceraldehyde are designated L, and stereoisomers related to D-glyceraldehyde are designated D. The func- tional groups of L-alanine are matched with those of L- glyceraldehyde by aligning those that can be intercon- verted 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 dextro- rotatory (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 am- 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, 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 de- scribes 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 oc- cur 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, in- cluding 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 diffi- cult 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, re- peating substructures in proteins (Chapter 4) generally requires that their constituent amino acids be of one stereochemical series. Cells are able to specifically syn- thesize 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 H CC H 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 H C COO� � CH3 N � H3 L-Alanine D-Alanine C (c) FIGURE 3–3 Stereoisomerism in �-amino acids. (a)The two stereoiso- mers of alanine, L- and D-alanine, are nonsuperimposable mirror im- ages 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 1CHO 3CH2OH H H C CHO CH2OH OH H3N � C COO� CH3 H H C 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 per- spective formulas, the carbons are lined up vertically, with the chiral atom in the center. The carbons in these molecules are numbered be- ginning 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: Amino Acids Can Be Classified by R Group Knowledge of the chemical properties of the common amino acids is central to an understanding of biochem- istry. 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 grada- tions 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 pro- tein structure by means of hydrophobic interactions. Glycine has the simplest structure. Although it is for- mally nonpolar, its very small side chain makes no real contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino acids, has a non- polar thioether group in its side chain. Proline has an Chapter 3 Amino Acids, Peptides, and Proteins78 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 Struc- ture 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: 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 tryp- tophan, with their aromatic side chains, are relatively nonpolar (hydrophobic). All can participate in hy- drophobic interactions. The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important func- tional 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 ex- tent phenylalanine, absorb ultraviolet light (Fig. 3–6; Box 3–1). This accounts for the characteristic strong ab- sorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the char- acterization 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 H 2N � H 2C 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 Aspartate GlutamateGlutamineAsparagine 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: 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 gluta- mine 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 mole- cules or residues are joined by a disulfide bond (Fig. 3–7). The disulfide-linked residues are strongly hy- drophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming co- valent links between parts of a protein molecule or be- tween two different polypeptide chains. Positively Charged (Basic) R Groups The most hydrophilic R groups are those that are either positively or nega- tively 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 � posi- tion on its aliphatic chain; arginine, which has a posi- tively 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 pro- ton 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 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-hydroxy