Amino Acids and the Role of pH (Lippincott Illustrated Reviews Biochemistry 8th Edition)

Amino Acids and the Role of pH 1 I. OVERVIEW Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of macromolecules. For example, enzymes and polypeptide hormones direct and re...

Amino Acids and the Role of pH 1 I. OVERVIEW Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of macromolecules. For example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and albumin, transport molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids and the importance of pH to normal protein and body function. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic functions. II. STRUCTURE Although more than 300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. These 20 standard amino acids are the only amino acids that are encoded by DNA, the genetic material in the cell. Nonstandard amino acids are produced by chemical modification of standard amino acids. Each amino acid has a carboxyl group, a primary amino group (except for proline, which has a secondary amino group), and a distinctive side chain or R group bonded to the α-carbon atom. At physiologic pH (∼7.4), the carboxyl group of an amino acid is dissociated, forming the negatively charged carboxylate ion (−COO−), and the amino group is protonated (−NH3+) (Fig. 1.1A). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, 17 in general, are not available for chemical reaction except for hydrogen bond or ionic bond formation (Fig. 1.1B). Amino acids within proteins are referred to as residues in reference to the residual structure remaining after peptide bond formation between consecutive amino acids within a peptide chain. It is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. Therefore, it is useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar, with an even distribution of electrons, or polar with an uneven distribution of electrons, such as acids and bases (Figs. 1.2 and 1.3). 18  Figure 1.1 A, B: Structural features of amino acids. A. Amino acids with nonpolar side chains Each of the amino acids in this category has a side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (see Fig. 1.2). The side chains of these amino acids can be thought of as “oily” or 19 lipid like, a property that promotes hydrophobic interactions (see Fig. 2.10). 1. Location in proteins: In proteins found in polar environments such as aqueous solutions, the side chains of nonpolar amino acids tend to cluster together in the interior of the protein (Fig. 1.4). This phenomenon is known as the hydrophobic effect and is the result of the hydrophobicity of the nonpolar R groups, which act much like droplets of oil that coalesce in an aqueous environment. By occupying the interior of the folded protein, these nonpolar R groups help give proteins their three-dimensional shape. Figure 1.2 Classification of the 20 standard amino acids, according to the charge and polarity of their side chains at acidic pH, is shown here and continues in Figure 1.3. Each amino acid is shown in its fully protonated form, with dissociable hydrogen ions represented in red. The pK values for the α-carboxyl and α-amino groups of the nonpolar amino acids are similar to those shown for glycine. 20  Figure 1.3 Classification of the 20 standard amino acids, according to the charge and polarity of their side chains at acidic pH (continued from Fig. 1.2). (Note: At physiologic pH (7.35 to 7.45), the α- carboxyl groups, the acidic side chains, and the side chain of free histidine are deprotonated.) For proteins located in a hydrophobic environment, such as within the hydrophobic core of a phospholipid membrane, nonpolar R groups are found on the outside surface of the protein, interacting with the lipid environment (see Fig. 1.4). The importance of these 21  hydrophobic interactions in stabilizing protein structure is discussed in Chapter 2. Figure 1.4 Location of nonpolar amino acids in soluble and membrane proteins. Sickle cell anemia, a disease that causes red blood cells to become sickle shaped rather than disc shaped, results from the replacement of polar glutamate with nonpolar valine at the sixth position in the β subunit of hemoglobin A (see Chapter 4). 2. Unique features of proline: Proline differs from other amino acids in that its side chain and α-amino nitrogen form a rigid, five-membered ring structure (Fig. 1.5). Proline, then, has a secondary (rather than a primary) amino group and is frequently referred to as an “imino acid.” The unique geometry of proline contributes to the formation of the extended fibrous structure of collagen (see Chapter 4, II Collagen B. Structure), but it interrupts the α-helices found in more compact globular proteins (see Chapter 2, III Secondary structure). 22  Figure 1.5 Comparison of the secondary amino group found in proline with the primary amino group found in other amino acids such as alanine. B. Amino acids with uncharged polar side chains These amino acids have zero net charge at physiologic pH of approximately 7.4, although the side chains of cysteine and tyrosine can lose a proton at an alkaline pH (see Fig. 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (Fig. 1.6). The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds. 1. Disulfide bond formation: The side chain of cysteine contains a sulfhydryl (thiol) group (−SH), which is an important component within the active site of many enzymes. In proteins, the –SH groups of two cysteines can be oxidized to form a covalent cross-link called a disulfide bond (−S–S–). Two cysteine residues that form a disulfide bond are referred to as cystine. (See Chapter 2 Section IV. B. for a further discussion of disulfide bond formation.) 23  Figure 1.6 Hydrogen bond between the phenolic hydroxyl group of tyrosine and another molecule containing a carbonyl group. Many extracellular proteins are stabilized by disulfide bonds. Albumin, a protein that functions in the transport of a variety of molecules in the blood, is one example. Fibrinogen, a blood protein converted to fibrin to stabilize blood clots, is another example. 2. Side chains as attachment sites for other compounds: The polar hydroxyl group of serine, threonine, and tyrosine can serve as a site of attachment for phosphate groups. Kinases are enzymes that catalyze phosphorylation reactions. Phosphatases are enzymes that remove the phosphate group. The changes in phosphorylation status of proteins (whether phosphorylated or not), especially of enzymes, alters their activation status; some enzymes are more active when phosphorylated while others are less active. In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins (see also Chapter 14 Section VII.). C. Amino acids with acidic side chains The amino acids aspartic acid and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, 24  containing a negatively charged carboxylate group (−COO−). The fully ionized forms are called aspartate and glutamate. D. Amino acids with basic side chains The side chains of the basic amino acids accept protons (see Fig. 1.3). At physiologic pH, the R groups of lysine and arginine are fully ionized and positively charged. In contrast, the free amino acid histidine is weakly basic and largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its R group can be either positively charged (protonated) or neutral, depending on the ionic environment provided by the protein. This important property of histidine contributes to the buffering role it plays in the functioning of proteins including hemoglobin (see Chapter 3). Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range (7.35 to 7.45). Clinical Application 1.1: Slower, Longer-Acting Insulin Created by Substituting Amino Acids Insulin glargine was first approved for use in the United States in the year 2000. It is a slower-acting form of insulin created in the laboratory by replacing the asparagine normally at position 21 on the A chain of insulin with glycine, and extending the carboxy terminus by two additional arginine residues. The result of these changes is a less water-soluble form of insulin with a net charge of +0.2, which is closer to 0, causing a slower absorption of insulin glargine from the site of injection. The glycine substitution prevents deamidation of the asparagine at acidic pH in the neutral, subcutaneous space. The additional arginine residues shift the isoelectric point from pH 5.4 to pH 6.7, making the molecule more soluble at acidic pH and less soluble at neutral pH. Insulin glargine is therefore a form of insulin that acts slowly, has longer activity, and requires less frequent injection. This form of insulin can be useful in the treatment of diabetes mellitus and help patients achieve better glycemic control. (See Chapter 23 for the structure of insulin.) E. Abbreviations and symbols for commonly occurring amino acids Each amino acid has an associated three-letter abbreviation and a one- letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules: 25  1. Unique first letter: If only one amino acid begins with a given letter, then that letter is used as its symbol. For example, V = valine. 2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine. 3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine. 4. Letter close to initial letter: For the remaining amino acids, a one- letter symbol is assigned that is close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine; Z is assigned to Glx, signifying either glutamic acid or glutamine; W is used for tryptophan and X is used to represent an unidentified amino acid. F. Amino acid isomers Because the α-carbon of an amino acid is attached to four different chemical groups, it is an asymmetric or chiral atom. Glycine is the exception because its α-carbon has two hydrogen substituents. Amino acids with a chiral α-carbon exist in two different isomeric forms, designated D and L, which are enantiomers, or mirror images (Fig. 1.8). (Note: Enantiomers are optically active. If an isomer, either D or L, causes the plane of polarized light to rotate clockwise, it is designated the [+] form.) All amino acids found in mammalian proteins are of the L configuration. However, D-amino acids are found in some antibiotics and in bacterial cell walls. (Note: Racemases enzymatically interconvert the D- and L-isomers of free amino acids.) 26 Figure 1.7 Abbreviations and symbols for the standard amino acids. 27