Biochemistry_Lippinco

Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. 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. 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


Biochemistry_Lippinco

Biochemistry_Lippinco. of amino acids. 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.


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II. STRUCTURE

Biochemistry_Lippinco. II. STRUCTURE


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Biochemistry_Lippinco. Although >300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. [Note: These standard amino acids are the only amino acids that are encoded by DNA, the genetic material in the cell (see p. 411). Nonstandard amino acids are produced by chemical modification of standard amino acids (see p. 45).] 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 (R group) bonded to the α carbon atom. At physiologic pH (~7.4), the carboxyl group 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, in general, are not available for chemical reaction except for hydrogen bond formation (Fig. 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role


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Biochemistry_Lippinco. peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Fig. 1.1B). Thus, 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 (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases) as shown in Figures 1.2 and 1.3.


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Biochemistry_Lippinco. 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.] A. Amino acids with nonpolar side chains Each of these amino acids has a nonpolar 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 lipid-like, a property that promotes hydrophobic interactions (see Fig. 2.10, p. 19).


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Biochemistry_Lippinco. 1. Location in proteins: In proteins found in aqueous solutions (a polar environment), the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Fig. 1.4). This phenomenon, known as the hydrophobic effect, 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 filling up the interior of the folded protein, these nonpolar R groups help give the protein its three-dimensional shape. However, for proteins that are located in a hydrophobic environment, such as a membrane, the 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 hydrophobic interactions in stabilizing protein structure is discussed on p. 19.


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Biochemistry_Lippinco. Sickle cell anemia, a disease of red blood cells that causes them 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 p. 36). 2. 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. It is frequently referred to as an “imino acid.” The unique geometry of proline contributes to the formation of the fibrous structure of collagen (see p. 45), but it interrupts the α-helices found in globular proteins (see p. 16). B. Amino acids with uncharged polar side chains


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Biochemistry_Lippinco. B. Amino acids with uncharged polar side chains These amino acids have zero net charge at physiologic pH, 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: The side chain of cysteine contains a sulfhydryl (thiol) group (−SH), which is an important component of 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 disulfide-linked cysteines are referred to as cystine. (See p. 19 for a further discussion of disulfide bond formation.)


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Biochemistry_Lippinco. Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions as a transporter for a variety of molecules, is an example. 2. Side chains as attachment sites for other compounds: The polar hydroxyl group of serine, threonine, and (rarely) tyrosine can serve as a site of attachment for structures such as a phosphate group. 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 p. 165). 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, containing a negatively charged carboxylate group (−COO−). The fully ionized forms are called aspartate and glutamate. D. Amino acids with basic side chains


Biochemistry_Lippinco

Biochemistry_Lippinco. 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 such proteins as hemoglobin (see p. 30). [Note: Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range.] E. Abbreviations and symbols for commonly occurring amino acids letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules. 1.


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Biochemistry_Lippinco. E. Abbreviations and symbols for commonly occurring amino acids letter symbol (Fig. 1.7). The one-letter codes are determined by the following rules. 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, or W = tryptophan (“twyptophan” as Elmer Fudd would say). 4.


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Biochemistry_Lippinco. 3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say). 4. Letter close to initial letter: For the remaining amino acids, a one-letter symbol is assigned that is as 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; and X is assigned to an unidentified amino acid. F. Amino acid isomers


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Biochemistry_Lippinco. F. Amino acid isomers Because the α-carbon of an amino acid is attached to four different chemical groups, it is an asymmetric (chiral) atom. Glycine is the exception because its α-carbon has two hydrogen substituents. Amino acids with a chiral αcarbon 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 (see p. 252). [Note: Racemases enzymatically interconvert the D-and L-isomers of free amino acids.] III. ACIDIC AND BASIC PROPERTIES


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Biochemistry_Lippinco. III. ACIDIC AND BASIC PROPERTIES Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as weak ionize to only a limited extent. The concentration of protons ([H+]) in aqueous solution is expressed as pH, where pH = log 1/[H+] or –log [H+]. The quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation. A. Equation derivation


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Biochemistry_Lippinco. A. Equation derivation Consider the release of a proton by a weak acid represented by HA: The salt or conjugate base, A−, is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is: [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A−. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.] By solving for the [H+] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by −1, and substituting pH = −log [H+] and pKa = −log Ka, we obtain the Henderson-Hasselbalch equation: B. Buffers


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Biochemistry_Lippinco. B. Buffers A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A−). If an acid such as HCl is added to a buffer, A− can neutralize it, being converted to HA in the process. If a base is added, HA can likewise neutralize it, being converted to A− in the process. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid-base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A− are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A− = CH3 – COO−) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution.


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Biochemistry_Lippinco. At pH values greater than the pKa, the deprotonated base form (CH3 – COO−) is the predominant species. C. Amino acid titration The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. 1. Carboxyl group dissociation: Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (Fig. 1.10). As the pH of the solution is raised, the −COOH group of form I can dissociate by donating a H+ to the medium. The release of a H+ results in the formation of the carboxylate group, −COO−. This structure is shown as form II, which is the dipolar form of the molecule (see Fig. 1.10). This form, also called a zwitterion (from the German word for “hybrid”), is the isoelectric form of alanine, that is, it has an overall (net) charge of zero. 2.


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Biochemistry_Lippinco. 2. Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K1, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: where I is the fully protonated form of alanine and II is the isoelectric form of alanine (see Fig. 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 3. Amino group dissociation: The second titratable group of alanine is the amino (−NH3+) group shown in Figure 1.10. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Fig. 1.10). 4.


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Biochemistry_Lippinco. 4. Alanine pKs: The sequential dissociation of H+ from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group. The pKa for the most acidic group (−COOH) is pK1, whereas the pKa for the next most acidic group (−NH3+) is pK2. [Note: The pKa of the α-carboxyl group of amino acids is ~2, whereas that of the α-amino group is ~9.] 5. Alanine titration curve: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: a.


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Biochemistry_Lippinco. Buffer pairs: The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3+/–NH2 pair can buffer in the region around pK2. b. When pH = pK: When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution. c.


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Biochemistry_Lippinco. c. Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) as shown in Figure 1.11. The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1).


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Biochemistry_Lippinco. Separation of plasma proteins by charge typically is done at a pH above the pI of the major proteins. Therefore, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobility pattern are suggestive of certain diseases. 6. Net charge at neutral pH: At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group (−NH3+), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes). D. Other applications of the Henderson-Hasselbalch equation


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Biochemistry_Lippinco. D. Other applications of the Henderson-Hasselbalch equation The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding salt form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO3−], and the carbon dioxide concentration [CO2] influence pH (Fig. 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Fig. 1.12B). Acidic drugs (HA) release a H+, causing a charged anion (A−) to form. Weak bases (BH+) can also release a H+. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B).


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Biochemistry_Lippinco. A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid, such as aspirin, the uncharged HA can permeate through membranes, but A− cannot. Likewise, for a weak base, such as morphine, the uncharged B form permeates through the cell membrane, but BH+ does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is determined by the relative concentrations of the charged (impermeant) and uncharged (permeant) forms. The ratio between the two forms is determined by the pH at the site of absorption and by the strength of the weak acid or base, which is represented by the pKa of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two compartments that differ in pH, for example, the stomach (pH 1.0–1.5) and blood plasma (pH 7.4). IV. CONCEPT MAPS


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Biochemistry_Lippinco. Students sometimes view biochemistry as a list of facts or equations to be memorized, rather than a body of concepts to be understood. Details provided to enrich understanding of these concepts inadvertently turn into distractions. What seems to be missing is a road map—a guide that provides the student with an understanding of how various topics fit together to “tell a story.” Therefore, in this text, a series of biochemical concept maps have been created to graphically illustrate relationships between ideas presented in a chapter and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualizing the connections between concepts. Material is represented in a hierarchic fashion, with the most inclusive, most general concepts at the top of the map, and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to


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Biochemistry_Lippinco. and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess. Concept map construction is described below.


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Biochemistry_Lippinco. A. Concept boxes and links Educators define concepts as “perceived regularities in events or objects.” In the biochemical maps, concepts include abstractions (for example, free energy), processes (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined concepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then drawn in boxes (Fig. 1.13A). The size of the type indicates the relative importance of each idea. Lines are drawn between concept boxes to show which are related. The label on the line defines the relationship between two concepts, so that it reads as a valid statement (that is, the connection creates meaning). The lines with arrowheads indicate in which direction the connection should be read (Fig. 1.14). B. Cross-links


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Biochemistry_Lippinco. B. Cross-links Unlike linear flow charts or outlines, concept maps may contain cross-links that allow the reader to visualize complex relationships between ideas represented in different parts of the map (Fig. 1.13B) or between the map and other chapters in this book (Fig. 1.13C). Cross-links can, thus, identify concepts that are central to more than one topic in biochemistry, empowering students to be effective in clinical situations and on the United States Medical Licensure Examination (USMLE) or other examinations that require integration of material. Students learn to visually perceive nonlinear relationships between facts, in contrast to cross-referencing within linear text. V. CHAPTER SUMMARY Each amino acid has an α-carboxyl group and a primary α-amino group (except for proline, which has a secondary amino group). At physiologic pH, the α-carboxyl group is dissociated, forming the negatively charged carboxylate ion (−COO−), and the α-amino group is protonated (−NH3+).


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Biochemistry_Lippinco. Each amino acid also contains one of 20 distinctive side chains attached to the α-carbon atom. The chemical nature of this R group determines the function of an amino acid in a protein and provides the basis for classification of the amino acids as nonpolar, uncharged polar, acidic (polar negative), or basic (polar positive). All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A−) is described by the Henderson-Hasselbalch equation. Buffering occurs within ±1 pH unit of the pKa and is maximal when pH = pKa, at which [A−] = [HA]. Because the α-carbon of each amino acid (except glycine) is attached to four different chemical groups, it is asymmetric (chiral), and amino acids exist in D-and L-isomeric forms that are optically active mirror images (enantiomers). The L-form of amino acids is found in proteins synthesized by the


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Biochemistry_Lippinco. it is asymmetric (chiral), and amino acids exist in D-and L-isomeric forms that are optically active mirror images (enantiomers). The L-form of amino acids is found in proteins synthesized by the human body.


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Biochemistry_Lippinco. Choose the ONE best answer. .1. Which one of the following statements concerning the titration curve for a nonpolar amino acid is correct? The letters A through D designate certain regions on the curve below. A. Point A represents the region where the amino acid is deprotonated. B. Point B represents a region of minimal buffering. C. Point C represents the region where the net charge on the amino acid is zero. D. Point D represents the pK of the amino acid’s carboxyl group. E. The amino acid could be lysine. Correct answer = C. Point C represents the isoelectric point, or pI, and as such is midway between pK1 and pK2 for a nonpolar amino acid. The amino acid is fully protonated at Point A. Point B represents a region of maximum buffering, as does Point D. Lysine is a basic amino acid, and free lysine has an ionizable side chain in addition to the ionizable α-amino and α-carboxyl groups.


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Biochemistry_Lippinco. .2. Which one of the following statements concerning the peptide shown below is correct?Val-Cys-Glu-Ser-Asp-Arg-Cys A. The peptide contains asparagine. B. The peptide contains a side chain with a secondary amino group. C. The peptide contains a side chain that can be phosphorylated. D. The peptide cannot form an internal disulfide bond. E. The peptide would move to the cathode (negative electrode) during electrophoresis at pH 5. Correct answer = C. The hydroxyl group of serine can accept a phosphate group. Asp is aspartate. Proline contains a secondary amino group. The two cysteine residues can, under oxidizing conditions, form a disulfide (covalent) bond. The net charge on the peptide at pH 5 is negative, and it would move to the anode.


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Biochemistry_Lippinco. .3. A 2-year-old child presents with metabolic acidosis after ingesting an unknown number of flavored aspirin tablets. At presentation, her blood pH was 7.0. Given that the pKa of aspirin (salicylic acid) is 3, calculate the ratio of its ionized to unionized forms at pH 7.0. Correct answer = 10,000 to 1. pH = pKa + log [A−]/[HA]. Therefore, 7 = 3 + × and × = 4. The ratio of A− (ionized) to HA (unionized), then, is 10,000 to 1 because the log of 10,000 is 4. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


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Biochemistry_Lippinco. I. OVERVIEW The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape that determines function. The complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels: primary, secondary, tertiary, and quaternary (Fig. 2.1). An examination of these hierarchies of increasing complexity has revealed that certain structural elements are repeated in a wide variety of proteins, suggesting that there are general rules regarding the ways in which proteins achieve their native, functional form. These repeated structural elements range from simple combinations of α-helices and β-sheets forming small motifs to the complex folding of polypeptide domains of multifunctional proteins (see p. 19). II. PRIMARY STRUCTURE


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Biochemistry_Lippinco. II. PRIMARY STRUCTURE The sequence of amino acids in a protein is called the primary structure of the protein. Understanding the primary structure of proteins is important because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease. A. Peptide bond


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Biochemistry_Lippinco. A. Peptide bond In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the α-carboxyl group of one amino acid and the αamino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Fig. 2.2). Peptide bonds are resistant to conditions that denature proteins, such as heating and high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated temperatures is required to break these bonds nonenzymically (see p. 14). 1.


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Biochemistry_Lippinco. 1. Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N-to the C-terminal end. For example, in Figure 2.2A, the order of the amino acids in the dipeptide is valine, alanine. Linkage of ≥50 amino acids through peptide bonds results in an unbranched chain called a polypeptide, or protein. Each component amino acid is called a residue because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a peptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valylglycylleucine. 2.


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Biochemistry_Lippinco. 2. Peptide bond characteristics: The peptide bond has a partial double-bond character, that is, it is shorter than a single bond and is rigid and planar (Fig. 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and the α-amino or α-carboxyl groups can be freely rotated (although they are limited by the size and character of the R groups). This allows the polypeptide chain to assume a variety of possible conformations. The peptide bond is almost always in the trans configuration (instead of the cis; see Fig. 2.2B), in large part because of steric interference of the R groups (side chains) when in the cis position. 3.


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Biochemistry_Lippinco. 3. Peptide bond polarity: Like all amide linkages, the −C = O and −NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The −C = O and −NH groups of the peptide bond are polar, however, and are involved in hydrogen bonds (for example, in α-helices and β-sheets), as described on pp. 16–17. B. Determining the amino acid composition of a polypeptide


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Biochemistry_Lippinco. The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anion-exchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them


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Biochemistry_Lippinco. the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin (a reagent that forms a purple compound with most amino acids, ammonia, and amines). The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer, an automated machine whose components are depicted in Figure 2.3.


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Biochemistry_Lippinco. C. Sequencing the peptide from its N-terminal end Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Fig. 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond such that it can be hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. Automated sequencers are now used. D. Cleaving the polypeptide into smaller fragments


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Biochemistry_Lippinco. D. Cleaving the polypeptide into smaller fragments Many polypeptides have a primary structure composed of >100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Fig. 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.] E. Determining a protein’s primary structure by DNA sequencing


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Biochemistry_Lippinco. E. Determining a protein’s primary structure by DNA sequencing The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, knowledge of the genetic code (see p. 447) allows the sequence of nucleotides to be translated into the corresponding amino acid sequence of that polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded chain and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification; see p. 459). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides. III. SECONDARY STRUCTURE


Biochemistry_Lippinco

Biochemistry_Lippinco. III. SECONDARY STRUCTURE The polypeptide backbone does not assume a random three-dimensional structure but, instead, generally forms regular arrangements of amino acids that are located near each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The α-helix, β-sheet, and βbend (or, β-turn) are examples of secondary structures commonly encountered in proteins. Each is stabilized by hydrogen bonds between atoms of the peptide backbone. [Note: The collagen α-chain helix, another example of secondary structure, is discussed on p. 45.] A. α-Helix


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Biochemistry_Lippinco. A. α-Helix Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a rigid, right-handed spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component L-amino acids extending outward from the central axis to avoid interfering sterically with each other (Fig. 2.6). A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, rigid, fibrous proteins whose structure is nearly entirely αhelical. They are a major component of tissues such as hair and skin. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule (see p. 26) found in muscles. 1.


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Biochemistry_Lippinco. 1. Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Fig. 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the –NH group of a peptide linkage four residues ahead in the polypeptide. This insures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix. 2. Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acids spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix. 3.


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Biochemistry_Lippinco. 3. Amino acids that disrupt an α-helix: The R group of an amino acid determines its propensity to be in an α-helix. Proline disrupts an α-helix because its rigid secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Glycine is also a “helix breaker” because its R group (a hydrogen) confers high flexibility. Additionally, amino acids with charged or bulky R groups (such as glutamate and tryptophan, respectively) and those with a branch at the β-carbon, the first carbon in the R group (for example, valine), have low α-helix propensity. B. β-Sheet


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Biochemistry_Lippinco. B. β-Sheet The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Fig. 2.7A). Because the surfaces of β-sheets appear “pleated,” they are often called βpleated sheets. [Note: Pleating results from successive α-carbons being slightly above or below the plane of the sheet.] Illustrations of protein structure often show β-strands as broad arrows (Fig. 2.7B). 1.


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Biochemistry_Lippinco. 1. Formation: A β-sheet is formed by two or more peptide chains (βstrands) aligned laterally and stabilized by hydrogen bonds between the carboxyl and amino groups of amino acids that either are far apart in a single polypeptide (intrachain bonds) or are in different polypeptide chains (interchain bonds). The adjacent β-strands are arranged either antiparallel to each other (with the N-termini alternating as shown in Fig. 2.7B) or parallel to each other (with the N-termini together as shown in Fig. 2.7C). On each β-strand, the R groups of adjacent amino acids extend in opposite directions, above and below the plane of the β-sheet. [Note: β-sheets are not flat and have a right-handed curl (twist) when viewed along the polypeptide backbone.] 2.


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Biochemistry_Lippinco. Comparing α-helices and β-sheets: In β-sheets, the β-strands are almost fully extended and the hydrogen bonds between the strands are perpendicular to the polypeptide backbone (see Fig. 2.7A). In contrast, in α-helices, the polypeptide is coiled and the hydrogen bonds are parallel to the backbone (see Fig. 2.6). The orientation of the R groups of the amino acid residues in both the αhelix and the β-sheet can result in formation of polar and nonpolar sides in these secondary structures, thereby making them amphipathic.


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Biochemistry_Lippinco. C. β-Bends (reverse turns, β-turns) β-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules and often include charged residues. [Note: β-Bends were given this name because they often connect successive strands of antiparallel βsheets.] β-Bends are generally composed of four amino acids, one of which may be proline, the amino acid that causes a kink in the polypeptide chain. Glycine, the amino acid with the smallest R group, is also frequently found in β-bends. β-Bends are stabilized by the formation of hydrogen bonds between the first and last residues in the bend. D. Nonrepetitive secondary structure


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Biochemistry_Lippinco. D. Nonrepetitive secondary structure Approximately one half of an average globular protein is organized into repetitive structures, such as the α-helix and β-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not random but rather simply have a less regular structure than those described above. [Note: The term “random coil” refers to the disordered structure obtained when proteins are denatured (see p. 20).] E. Supersecondary structures (motifs)


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Biochemistry_Lippinco. E. Supersecondary structures (motifs) Globular proteins are constructed by combining secondary structural elements (that is, α-helices, β-sheets, and coils), producing specific geometric patterns, or motifs. These form primarily the core (interior) region of the molecule. They are connected by loop regions (for example, β-bends) at the surface of the protein. Supersecondary structures are usually produced by the close packing of side chains from adjacent secondary structural elements. For example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8. Motifs may be associated with particular functions. Proteins that bind to DNA contain a limited number of motifs. The helix–loop–helix motif is an example found in a number of proteins that function as transcription factors (see p. 438). IV. TERTIARY STRUCTURE


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. TERTIARY STRUCTURE The primary structure of a polypeptide chain determines its tertiary structure. “Tertiary” refers both to the folding of domains (the basic units of structure and function; see A. below) and to the final arrangement of domains in the polypeptide. The tertiary structure of globular proteins in aqueous solution is compact, with a high density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule. A. Domains


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Biochemistry_Lippinco. A. Domains Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are >200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain. B. Stabilizing interactions The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins.


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Biochemistry_Lippinco. 1. Disulfide bonds: A disulfide bond (–S–S–) is a covalent linkage formed from the sulfhydryl group (−SH) of each of two cysteine residues to produce a cystine residue (Fig. 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide or may even be located on two different polypeptides. The folding of the polypeptide(s) brings the cysteine residues into proximity and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells. [Note: Protein disulfide isomerase breaks and reforms disulfide bonds during folding.] 2.


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Biochemistry_Lippinco. Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Fig. 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Fig. 1.4, p. 4).] In each case, a segregation of R groups occurs that is energetically most favorable. 3.


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Biochemistry_Lippinco. 3. Hydrogen bonds: Amino acid side chains containing oxygen-or nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Fig. 2.11; see also Fig. 1.6, p. 4). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein. 4. Ionic interactions: Negatively charged groups, such as the carboxylate group (−COO−) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (−NH3+) in the side chain of lysine (see Fig. 2.11). C. Protein folding


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Biochemistry_Lippinco. C. Protein folding Interactions between the side chains of amino acids determine how a linear polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, involves nonrandom, ordered pathways. As a peptide folds, secondary structures form, driven by the hydrophobic effect (that is, hydrophobic groups come together as water is released). These small structures combine to form larger structures. Additional events stabilize secondary structure and initiate formation of tertiary structure. In the last stage, the peptide achieves its fully folded, native (functional) form characterized by a low-energy state (Fig. 2.12). [Note: Some biologically active proteins or segments thereof lack a stable tertiary structure. They are referred to as intrinsically disordered proteins.] D. Protein denaturation


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Biochemistry_Lippinco. D. Protein denaturation Denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, urea, organic solvents, strong acids or bases, detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins remain permanently disordered once denatured. Denatured proteins are often insoluble and precipitate from solution. E. Chaperones in protein folding


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Biochemistry_Lippinco. The information needed for correct protein folding is contained in the primary structure of the polypeptide. However, most denatured proteins do not resume their native conformations even under favorable environmental conditions. This is because, for many proteins, folding is a facilitated process that requires a specialized group of proteins, referred to as molecular chaperones, and ATP hydrolysis. The chaperones, also known as heat shock proteins (HSP), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed (for example, Hsp70). Others form cage-like macromolecular structures composed of two stacked rings. The partially folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage-like chaperones are sometimes


Biochemistry_Lippinco

Biochemistry_Lippinco. folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage-like chaperones are sometimes referred to as chaperonins.] Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation-prone hydrophobic regions in nascent (and denatured) polypeptides, preventing premature folding.


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Biochemistry_Lippinco. V. QUATERNARY STRUCTURE Many proteins consist of a single polypeptide chain and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions). Subunits either may function independently of each other or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see p. 29). Isoforms are proteins that perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isozymes (see p. 65).


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Biochemistry_Lippinco. VI. PROTEIN MISFOLDING Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 247). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of misfolded proteins are associated with a number of diseases. A. Amyloid diseases


Biochemistry_Lippinco

Biochemistry_Lippinco. Misfolding of proteins may occur spontaneously or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformation that leads to the spontaneous formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble fibrous protein aggregates, called amyloids, has been implicated in neurodegenerative disorders such as Parkinson disease and Alzheimer disease (AD). The dominant component of the amyloid plaque that accumulates in AD is amyloid β (Aβ), an extracellular peptide containing 40–42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic βpleated sheet secondary structure in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic


Biochemistry_Lippinco

Biochemistry_Lippinco. in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The Aβ that is deposited in the brain in AD is derived by enzymic cleavages (by secretases) from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues (Fig. 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of AD are not genetically based, although at least 5% of cases are familial. A second biologic factor involved in the development of AD is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears


Biochemistry_Lippinco

Biochemistry_Lippinco. fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears to block the actions of its normal counterpart. [Note: In Parkinson disease, amyloid is formed from α-synuclein protein.]


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Biochemistry_Lippinco. B. Prion (proteinaceous infectious particle) diseases


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Biochemistry_Lippinco. The prion protein (PrP) is the causative agent of transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called “mad cow” disease). After an extensive series of purification procedures, scientists were surprised to find that the infectivity of the agent causing scrapie in sheep was associated with a single protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPSc (Sc = scrapie). It is highly resistant to proteolytic degradation and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrPC (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational


Biochemistry_Lippinco

Biochemistry_Lippinco. as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrPC. Research has demonstrated that a number of αhelices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Fig. 2.14). This conformational difference is presumably what confers relative resistance to proteolytic degradation of infectious prions and permits them to be distinguished from the normal PrPC in infected tissue. The infective agent is, thus, an altered version of a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSE are invariably fatal, and no treatment is currently available that can alter this


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Biochemistry_Lippinco. a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSE are invariably fatal, and no treatment is currently available that can alter this outcome.


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VII. CHAPTER SUMMARY

Biochemistry_Lippinco. VII. CHAPTER SUMMARY


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Biochemistry_Lippinco. Central to understanding protein structure is the concept of the native conformation (Fig. 2.15), which is the functional, fully folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a specialized group of proteins named chaperones is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation


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Biochemistry_Lippinco. which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease (AD) and the transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease. In AD, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets. In TSE, the infective agent is an altered version of a normal prion protein that acts as a template for converting normal protein to the pathogenic conformation.


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Biochemistry_Lippinco. Choose the ONE best answer. .1. Which one of the following statements concerning protein structure is correct? A. Proteins consisting of one polypeptide have quaternary structure that is stabilized by covalent bonds. B. The peptide bonds that link amino acids in a protein most commonly occur in the cis configuration. C. The formation of a disulfide bond in a protein requires the participating cysteine residues to be adjacent in the primary structure. D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix. E. The primary driving force for protein folding is the hydrophobic effect.


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Biochemistry_Lippinco. D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix. E. The primary driving force for protein folding is the hydrophobic effect. Correct answer = E. The hydrophobic effect, or the tendency of nonpolar entities to associate in a polar environment, is the primary driving force of protein folding. Quaternary structure requires more than one polypeptide, and, when present, it is stabilized primarily by noncovalent bonds. The peptide bond is almost always trans. The two cysteine residues participating in disulfide bond formation may be a great distance apart in the amino acid sequence of a polypeptide (or on two separate polypeptides) but are brought into close proximity by the three-dimensional folding of the polypeptide. Denaturation may be reversible or irreversible.


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Biochemistry_Lippinco. .2. A particular point mutation results in disruption of the α-helical structure in a segment of the mutant protein. The most likely change in the primary structure of the mutant protein is: A. glutamate to aspartate. B. lysine to arginine. C. methionine to proline. D. valine to alanine. Correct answer = C. Proline, because of its secondary amino group, is incompatible with an α-helix. Glutamate, aspartate, lysine, and arginine are charged amino acids, and valine is a branched amino acid. Charged and branched (bulky) amino acids may disrupt an α-helix. [Note: The flexibility of glycine’s R group can also disrupt an α-helix.] .3. In comparing the α-helix to the β-sheet, which statement is correct only for the β-sheet? A. Extensive hydrogen bonds between the carbonyl oxygen (C=O) and the amide hydrogen (N−H) of the peptide bond are formed. B. It may be found in typical globular proteins. C. It is stabilized by interchain hydrogen bonds.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. It may be found in typical globular proteins. C. It is stabilized by interchain hydrogen bonds. D. It is an example of secondary structure. E. It may be found in supersecondary structures. Correct answer = C. The β-sheet is stabilized by interchain hydrogen bonds formed between separate polypeptide chains and by intrachain hydrogen bonds formed between regions of a single polypeptide. The α-helix, however, is stabilized only by intrachain hydrogen bonds. Statements A, B, D, and E are true for both of these secondary structural elements. .4. An 80-year-old man presented with impairment of intellectual function and alterations in behavior. His family reported progressive disorientation and memory loss over the last 6 months. There is no family history of dementia. The patient was tentatively diagnosed with Alzheimer disease (AD). Which one of the following best describes AD? A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence.


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Biochemistry_Lippinco. A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence. B. It results from accumulation of denatured proteins that have random conformations. C. It is associated with the accumulation of amyloid precursor protein. D. It is associated with the deposition of neurotoxic amyloid β peptide aggregates. E. It is an environmentally produced disease not influenced by the genetics of the individual. F. It is caused by the infectious β-sheet form of a host-cell protein.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. It is an environmentally produced disease not influenced by the genetics of the individual. F. It is caused by the infectious β-sheet form of a host-cell protein. Correct answer = D. Alzheimer disease (AD) is associated with long, fibrillar protein assemblies consisting of β-pleated sheets found in the brain and elsewhere. The disease is associated with abnormal processing of a normal protein. The accumulated altered protein occurs in a β-pleated sheet conformation that is neurotoxic. The amyloid β that is deposited in the brain in AD is derived by proteolytic cleavages from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues. Most cases of AD are sporadic, although at least 5% of cases are familial. Prion diseases, such as Creutzfeldt-Jakob, are caused by the infectious β-sheet form (PrPSc) of a host-cell protein (PrPC).


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Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW The previous chapter described the types of secondary and tertiary structures that are the bricks and mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4. II. GLOBULAR HEMEPROTEINS


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Biochemistry_Lippinco. II. GLOBULAR HEMEPROTEINS Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 75). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2). A. Heme structure


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Heme structure Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+), as shown in Figure 3.1. The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind O2 (Fig. 3.2). (See pp. 278 and 282, respectively, for a discussion of heme synthesis and degradation.) B. Myoglobin structure and function


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Biochemistry_Lippinco. B. Myoglobin structure and function Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as an oxygen reservoir and as an oxygen carrier that increases the rate of oxygen transport within the muscle cell. [Note: Surprisingly, mouse myoglobin double knockouts (see p. 502) have an apparently normal phenotype.] Myoglobin consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin. 1.


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Biochemistry_Lippinco. 1. α-Helical content: Myoglobin is a compact molecule, with ~80% of its polypeptide chain folded into eight stretches of α-helix. These α-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix (see p. 16) or by β-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 19). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2. Location of polar and nonpolar amino acid residues: The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds, both with each other and with water. 3.


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Biochemistry_Lippinco. 3. Binding of the heme group: The heme group of the myoglobin molecule sits in a crevice, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (see Fig. 3.2B). One, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+ . Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by Fe2+ (oxidation to the ferric [Fe3+] form) occurs only rarely. C. Hemoglobin structure and function


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Hemoglobin structure and function Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions (Fig. 3.3). Each chain (subunit) has stretches of αhelical structure and a hydrophobic heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport protons (H+) and carbon dioxide (CO2) from the tissues to the lungs and can carry four molecules of O2 from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).


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Biochemistry_Lippinco. Obtaining O2 from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because O2 is only slightly soluble in aqueous solutions such as blood.


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Biochemistry_Lippinco. 1. Quaternary structure: The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions (Fig. 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule but also in a region on the surface of each subunit. Multiple interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.] In contrast, the two dimers are held together primarily by polar bonds. The weaker interactions between the dimers allow them to move with respect to one other. This movement results in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Fig. 3.4). a.


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Biochemistry_Lippinco. a. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T conformation is the low-oxygen-affinity form of hemoglobin. b. R form: The binding of O2 to hemoglobin causes the rupture of some of the polar bonds between the two αβ dimers, allowing movement. Specifically, the binding of O2 to the heme Fe2+ pulls the iron into the plane of the heme (Fig. 3.5). Because the iron is also linked to the proximal histidine (F8), the resulting movement of the globin chains alters the interface between the αβ dimers. This leads to a structure called the “R,” or relaxed form (see Fig. 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin. oxygen (O2) is not bound. B. Into the plane of the heme upon O2 binding. D. Oxygen binding to myoglobin and hemoglobin


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Biochemistry_Lippinco. Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full), as shown in Figure 3.6. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the oxygen saturation of arterial blood based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin.] 1. Oxygen-dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. [Note: pO2 may also be represented as PO2.] The curves for myoglobin and hemoglobin show important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half


Biochemistry_Lippinco

Biochemistry_Lippinco. important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly O2 binds), the lower the P50.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Fig. 3.6). This reflects the fact that myoglobin reversibly binds a single molecule of O2. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:


Biochemistry_Lippinco

Biochemistry_Lippinco. The equilibrium is shifted to the right or to the left as O2 is added to or removed from the system. [Note: Myoglobin is designed to bind O2 released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases O2 within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Fig. 3.6), indicating that the subunits cooperate in binding O2. Cooperative binding of O2 by the four subunits of hemoglobin means that the binding of an oxygen molecule at one subunit increases the oxygen affinity of the remaining subunits in the same hemoglobin tetramer (Fig. 3.7). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen molecules occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Fig. 3.6). E. Allosteric effectors


Biochemistry_Lippinco

Biochemistry_Lippinco. The ability of hemoglobin to reversibly bind O2 is affected by the pO2, the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the tetrameric hemoglobin molecule causes structural changes that affect the binding of O2 to the heme iron at other sites on the molecule. [Note: The binding of O2 to monomeric myoglobin is not influenced by allosteric effectors.] 1. Oxygen: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one subunit and transmitted to other subunits in the hemoglobin tetramer. The net effect of this cooperativity is that the affinity of hemoglobin for the last oxygen molecule bound is ~300 times greater than its affinity for the first oxygen molecule bound. Oxygen, then, is an allosteric effector of hemoglobin. It stabilizes the R form.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Loading and unloading oxygen: The cooperative binding of O2 allows hemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2. This can be seen in Figure 3.6, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, oxygen concentration is high, and hemoglobin becomes virtually saturated (or “loaded”) with O2. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its O2 for use in the oxidative metabolism of the tissues (Fig. 3.8).


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Biochemistry_Lippinco. b. Significance of the sigmoidal oxygen-dissociation curve: The steep slope of the oxygen-dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver O2 efficiently from sites of high to sites of low pO2. A molecule with a hyperbolic oxygen-dissociation curve, such as myoglobin, could not achieve the same degree of O2 release within this range of pO2. Instead, it would have maximum affinity for O2 throughout this oxygen pressure range and, therefore, would deliver no O2 to the tissues.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Bohr effect: The release of O2 from hemoglobin is enhanced when the pH is lowered (proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Fig. 3.9). Both, then, stabilize the T (deoxy) form. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater oxygen affinity, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. a. Source of the protons that lower pH: The concentration of both H+ and


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Source of the protons that lower pH: The concentration of both H+ and CO2 in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air. In the tissues, CO2 is converted by zinc-containing carbonic anhydrase to carbonic acid: which spontaneously loses a H+, becoming bicarbonate (the major blood buffer): The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (that is, lungs having a higher pH and tissues a lower pH) favors the unloading of O2 in the peripheral tissues and the loading of O2 in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of O2.


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Biochemistry_Lippinco. b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for H+ than does oxyhemoglobin. This is caused by ionizable groups such as specific histidine side chains that have a higher pKa (see p. 6) in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of H+ (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is an important blood buffer.] The Bohr effect can be represented schematically as: where an increase in H+ (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in H+) shifts the equilibrium to the left.


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Biochemistry_Lippinco. 3. 2,3-BPG effect on oxygen affinity: 2,3-BPG is an important regulator of the binding of O2 to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Fig. 3.10; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis). a. 2,3-BPG binding to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the T conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as: b.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released. c. Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.


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Biochemistry_Lippinco. d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).


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Biochemistry_Lippinco. e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:


Biochemistry_Lippinco

Biochemistry_Lippinco. The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. CO binding: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin. When CO binds to one or more of the four heme sites, hemoglobin shifts to the R conformation, causing the remaining heme sites to bind O2 with high affinity. This shifts the oxygen-dissociation curve to the left and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release O2 to the tissues (Fig. 3.13). [Note: The affinity of hemoglobin for CO is 220 times greater than for O2. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood. For example, increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the


Biochemistry_Lippinco

Biochemistry_Lippinco. from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, CO2, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBC, thereby modulating NO availability and influencing vessel diameter.


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Biochemistry_Lippinco. F. Minor hemoglobins It is important to remember that human hemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Fig. 3.14). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α-like) polypeptides and two βglobin (or β-like) polypeptides. Certain hemoglobins, such as HbF, are normally synthesized only during fetal development, whereas others, such as HbA2, are synthesized in the adult, although at low levels compared with HbA. HbA can also become modified by the covalent addition of a hexose (see 3. below). 1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Fig. 3.14). The γ chains are members of the β-globin gene family (see p. 34).


Biochemistry_Lippinco

Biochemistry_Lippinco. a. HbF synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF. HbF is the major hemoglobin found in the fetus and newborn, accounting for ~60% of the total hemoglobin in the RBC during the last months of fetal life (Fig. 3.15). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. Figure 3.15 shows the relative production of each type of hemoglobin chain during fetal and postnatal life. [Note: HbF represents <2% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only


Biochemistry_Lippinco

Biochemistry_Lippinco. hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only weakly binding 2,3-BPG. [Note: The γ-globin chains of HbF lack some of the positively charged amino acids that are responsible for binding 2,3BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the oxygen affinity of hemoglobin, the weaker interaction between 2,3BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. In contrast, if both HbA and HbF are stripped of their 2,3-BPG, they then have a similar oxygen affinity. The higher oxygen affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to the RBC of the fetus.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting ~2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Fig. 3.14). 3. Hemoglobin A1c: Under physiologic conditions, HbA is slowly glycated (nonenzymically condensed with a hexose), the extent of glycation being dependent on the plasma concentration of the hexose. The most abundant form of glycated hemoglobin is HbA1c. It has glucose residues attached predominantly to the amino groups of the N-terminal valines of the βglobin chains (Fig. 3.16). Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of HbA1c levels in assessing average blood glucose levels in patients with diabetes.) III. GLOBIN GENE ORGANIZATION


Biochemistry_Lippinco

Biochemistry_Lippinco. III. GLOBIN GENE ORGANIZATION To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobin, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed. A. α-Gene family The genes coding for the α-globin and β-globin subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Fig. 3.17). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as an α-globin-like component of embryonic hemoglobin. [Note: Globin gene families also contain globinlike genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.] B. β-Gene family


Biochemistry_Lippinco

Biochemistry_Lippinco. B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Fig. 3.17). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in HbF), and the δ gene that codes for the globin chain found in the minor adult hemoglobin HbA2. C. Steps in globin chain synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The ribonucleic acid (RNA) produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.18. A more detailed description of gene expression is presented in Unit VII, Chapters 30–32.) IV. HEMOGLOBINOPATHIES


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. HEMOGLOBINOPATHIES Hemoglobinopathies are defined as a group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (HbS), hemoglobin C disease (HbC), hemoglobin SC disease (HbS + HbC = HbSC), and the thalassemias are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy). A. Sickle cell anemia (hemoglobin S disease)


Biochemistry_Lippinco

Biochemistry_Lippinco. Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder caused by a single nucleotide substitution (a point mutation, see p. 449) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 50,000 Americans. It occurs primarily in the African American population, affecting 1 in 500 newborn African American infants. Sickle cell anemia is an autosomalrecessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS2, is referred to as HbS.] An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p.


Biochemistry_Lippinco

Biochemistry_Lippinco. by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in infancy. [Note: The lifetime of a RBC in sickle cell anemia is <20 days, compared with 120 days for normal RBC, hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Life expectancy is reduced. Heterozygotes, representing 1 in 12 African Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both HbS and HbA, and these individuals have sickle cell trait. They usually do not show clinical symptoms (but may under conditions of extreme physical exertion with dehydration) and can have a normal life span.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Amino acid substitution in HbS β chains:: A molecule of HbS contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Fig. 3.19). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Fig. 3.20). This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA. [Note: Electrophoresis of hemoglobin obtained from lysed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell anemia (or, sickle cell disease). DNA analysis also is used (see p. 493).] 2.


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Biochemistry_Lippinco. Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Fig. 3.21). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of O2 leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually ischemic death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS. [Note: The mean diameter of RBC is 7.5 µm, whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through


Biochemistry_Lippinco

Biochemistry_Lippinco. whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through small vessels difficult, thereby causing microvascular occlusion.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Variables that increase sickling: The extent of sickling and, therefore, the severity of disease are enhanced by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the oxygen affinity of HbS). These variables include decreased pO2, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in RBC. 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed RBC reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload that can result in hemosiderosis (see p. 404), bloodborne infections, and immunologic complications. Hydroxyurea (hydroxycarbamide), an antitumor drug, is therapeutically useful because it increases circulating levels of HbF, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. Stem cell transplantation is possible. [Note: The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child.] 5.


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Biochemistry_Lippinco. Possible selective advantage of the heterozygous state: The high frequency of the βS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to the severe malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for HbS, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. For example, in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria. B. Hemoglobin C disease


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Hemoglobin C disease Like HbS, HbC is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain (see Fig. 3.19). In HbC, however, a lysine is substituted for the glutamate (as compared with a valine substitution in HbS). [Note: This substitution causes HbC to move more slowly toward the anode than HbA or HbS does (see Fig. 3.20).] Rare patients homozygous for HbC generally have a relatively mild, chronic hemolytic anemia. They do not suffer from infarctive crises, and no specific therapy is required. C. Hemoglobin SC disease


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Biochemistry_Lippinco. C. Hemoglobin SC disease HbSC disease is another of the RBC sickling diseases. In this disease, some β-globin chains have the sickle cell mutation, whereas other β-globin chains carry the mutation found in HbC disease. [Note: Patients with HbSC disease are doubly heterozygous. They are called compound heterozygotes because both of their β-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in HbSC disease than in sickle cell anemia and may even be at the low end of the normal range. The clinical course of adults with HbSC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe. However, there is significant clinical variability. D. Methemoglobinemias