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Cell_Biology_Alberts. The present database of known protein sequences contains more than twenty million entries, and it is growing very rapidly as more and more genomes are sequenced—revealing huge numbers of new genes that encode proteins. The encoded polypeptides range widely in size, from 6 amino acids to a gigantic protein of 33,000 amino acids. Protein comparisons are important because related structures often imply related functions. Many years of experimentation can be saved by discovering that a new protein has an amino acid sequence similarity with a protein of known function. Such sequence relationships, for example, first indicated that certain genes that cause mammalian cells to become cancerous encode protein kinases (discussed in Chapter 20).


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Cell_Biology_Alberts. As previously stated, most proteins are composed of a series of protein domains, in which different regions of the polypeptide chain fold independently to form compact structures. Such multidomain proteins are believed to have originated from the accidental joining of the DNA sequences that encode each domain, creating a new gene. In an evolutionary process called domain shuffling, many large proteins have evolved through the joining of preexisting domains in new combinations (Figure 3–14). Novel binding surfaces have often been created at the juxtaposition of domains, and many of the functional sites where proteins bind to small molecules are found to be located there. A subset of protein domains has been especially mobile during evolution; these seem to have particularly versatile structures and are sometimes referred to as protein modules. The structure of one, the SH2 domain, was illustrated in Figure 3–6. Three other abundant protein domains are illustrated in Figure 3–15.


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Cell_Biology_Alberts. Each of the domains shown has a stable core structure formed from strands of β sheets, from which less-ordered loops of polypeptide chain protrude. The loops are ideally situated to form binding sites for other molecules, as most clearly demonstrated for the immunoglobulin fold, which forms the basis for antibody molecules. Such β-sheet-based domains may have achieved their evolutionary success because they provide a convenient framework for the generation of new binding sites for ligands, requiring only small changes to their protruding loops (see Figure 3–42).


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Cell_Biology_Alberts. Figure 3–14 Domain shuffling. An extensive shuffling of blocks of protein sequence (protein domains) has occurred during protein evolution. Those portions of a protein denoted by the same shape and color in this diagram are evolutionarily related. Serine proteases like chymotrypsin are formed from two domains (brown). In the three other proteases shown, which are highly regulated and more specialized, these two protease domains are connected to one or more domains that are similar to domains found in epidermal growth factor (EGF; green), to a calcium-binding protein (yellow), or to a “kringle” domain (blue). Chymotrypsin is illustrated in Figure 3–12. Figure 3–15 The three-dimensional structures of three commonly used protein domains. In these ribbon diagrams, β-sheet strands are shown as arrows, and the Nand C-termini are indicated by red spheres. Many more such “modules” exist in nature. (Adapted from


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Cell_Biology_Alberts. M. Baron, D.G. Norman and I.D. Campbell, Trends Biochem. Sci. 16:13–17, 1991, with permission from Elsevier, and D.J. Leahy et al., Science 258:987–991, 1992, with permission from AAAS.) Figure 3–16 an extended structure formed from a series of protein domains. Four fibronectin type 3 domains (see Figure 3–15) from the extracellular matrix molecule fibronectin are illustrated in (A) ribbon and (B) space-filling models. (Adapted from D.J. Leahy, I. Aukhil and H.P. Erickson, Cell 84:155–164, 1996. With permission from Elsevier.)


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Cell_Biology_Alberts. A second feature of these protein domains that explains their utility is the ease with which they can be integrated into other proteins. Two of the three domains illustrated in Figure 3–15 have their Nand C-terminal ends at opposite poles of the domain. When the DNA encoding such a domain undergoes tandem duplication, which is not unusual in the evolution of genomes (discussed in Chapter 4), the duplicated domains with this “in-line” arrangement can be readily linked in series to form extended structures—either with themselves or with other in-line domains (Figure 3–16). Stiff extended structures composed of a series of domains are especially common in extracellular matrix molecules and in the extracellular portions of cell-surface receptor proteins. Other frequently used domains, including the kringle domain illustrated in Figure 3–15 and the SH2 domain, are of a “plug-in” type, with their Nand C-termini close together. After genomic rearrangements, such domains are usually


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Cell_Biology_Alberts. including the kringle domain illustrated in Figure 3–15 and the SH2 domain, are of a “plug-in” type, with their Nand C-termini close together. After genomic rearrangements, such domains are usually accommodated as an insertion into a loop region of a second protein.


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Cell_Biology_Alberts. A comparison of the relative frequency of domain utilization in different eukaryotes reveals that, for many common domains, such as protein kinases, this frequency is similar in organisms as diverse as yeast, plants, worms, flies, and humans. But there are some notable exceptions, such as the Major Histocompatibility Complex (MHC) antigen-recognition domain (see Figure 24–36) that is present in 57 copies in humans, but absent in the other four organisms just mentioned. Domains such as these have specialized functions that are not shared with the other eukaryotes; they are assumed to have been strongly selected for during recent evolution to produce the multiple copies observed. Similarly, the SH2 domain shows an unusual increase in its numbers in higher eukaryotes; such domains might be assumed to be especially useful for multicellularity. Certain Pairs of Domains Are Found Together in Many Proteins


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Cell_Biology_Alberts. Certain Pairs of Domains Are Found Together in Many Proteins We can construct a large table displaying domain usage for each organism whose genome sequence is known. For example, the human genome contains the DNA sequences for about 1000 immunoglobulin domains, 500 protein kinase domains, 250 DNA-binding homeodomains, 300 SH3 domains, and 120 SH2 domains. In addition, we find that more than two-thirds of all proteins consist of two or more domains, and that the same pairs of domains occur repeatedly in the same relative arrangement in a protein. Although half of all domain families are common to archaea, bacteria, and eukaryotes, only about 5% of the two-domain combinations are similarly shared. This pattern suggests that most proteins containing especially useful two-domain combinations arose through domain shuffling relatively late in evolution. The Human Genome Encodes a Complex Set of Proteins, Revealing That Much Remains Unknown


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Cell_Biology_Alberts. The Human Genome Encodes a Complex Set of Proteins, Revealing That Much Remains Unknown The result of sequencing the human genome has been surprising, because it reveals that our chromosomes contain only about 21,000 protein-coding genes. Based on this number alone, we would appear to be no more complex than the tiny mustard weed, Arabidopsis, and only about 1.3-fold more complex than a nematode worm. The genome sequences also reveal that vertebrates have inherited nearly all of their protein domains from invertebrates—with only 7% of identified human domains being vertebrate-specific. Each of our proteins is on average more complicated, however (Figure 3–17). Domain shuffling during vertebrate evolution has given rise to many novel


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Cell_Biology_Alberts. Each of our proteins is on average more complicated, however (Figure 3–17). Domain shuffling during vertebrate evolution has given rise to many novel Figure 3–17 Domain structure of a group of evolutionarily related proteins that are thought to have a similar function. In general, there is a tendency for the proteins in more complex organisms, such as humans, to contain additional domains—as is the case for the DNA-binding protein compared here. combinations of protein domains, with the result that there are nearly twice as many combinations of domains found in human proteins as in a worm or a fly. Thus, for example, the trypsinlike serine protease domain is linked to at least 18 other types of protein domains in human proteins, whereas it is found covalently joined to only 5 different domains in the worm. This extra variety in our proteins greatly increases the range of protein–protein interactions possible (see Figure 3–79), but how it contributes to making us human is not known.


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Cell_Biology_Alberts. The complexity of living organisms is staggering, and it is quite sobering to note that we currently lack even the tiniest hint of what the function might be for more than 10,000 of the proteins that have thus far been identified through examining the human genome. There are certainly enormous challenges ahead for the next generation of cell biologists, with no shortage of fascinating mysteries to solve.


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Cell_Biology_Alberts. The same weak noncovalent bonds that enable a protein chain to fold into a specific conformation also allow proteins to bind to each other to produce larger structures in the cell. Any region of a protein’s surface that can interact with another molecule through sets of noncovalent bonds is called a binding site. A protein can contain binding sites for various large and small molecules. If a binding site recognizes the surface of a second protein, the tight binding of two folded polypeptide chains at this site creates a larger protein molecule with a precisely defined geometry. Each polypeptide chain in such a protein is called a protein subunit.


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Cell_Biology_Alberts. In the simplest case, two identical folded polypeptide chains bind to each other in a “head-to-head” arrangement, forming a symmetric complex of two protein subunits (a dimer) held together by interactions between two identical binding sites. The Cro repressor protein—a viral gene regulatory protein that binds to DNA to turn specific viral genes off in an infected bacterial cell—provides an example (Figure 3–18). Cells contain many other types of symmetric protein complexes, formed from multiple copies of a single polypeptide chain (for example, see Figure 3–20 below). Many of the proteins in cells contain two or more types of polypeptide chains. Hemoglobin, the protein that carries oxygen in red blood cells, contains two identical α-globin subunits and two identical β-globin subunits, symmetrically arranged (Figure 3–19). Such multisubunit proteins are very common in cells, and they can be very large (Movie 3.6).


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Cell_Biology_Alberts. Most of the proteins that we have discussed so far are globular proteins, in which the polypeptide chain folds up into a compact shape like a ball with an irregular surface. Some of these protein molecules can nevertheless assemble to form filaments that may span the entire length of a cell. Most simply, a long chain of identical protein molecules can be constructed if each molecule has a binding Figure 3–18 Two identical protein subunits binding together to form a symmetric protein dimer. The Cro repressor protein from bacteriophage lambda binds to DNA to turn off a specific subset of viral genes. Its two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region). (Adapted from D.H. Ohlendorf, D.E. Tronrud and B.W. Matthews, J. Mol. Biol. 280:129–136, 1998. With permission from Academic Press.)


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Cell_Biology_Alberts. D.E. Tronrud and B.W. Matthews, J. Mol. Biol. 280:129–136, 1998. With permission from Academic Press.) Figure 3–19 a protein formed as a symmetric assembly using two each of two different subunits. Hemoglobin is an abundant protein in red blood cells that contains two copies of α-globin (green) and two copies of β-globin (blue). Each of these four polypeptide chains contains a heme molecule (red), which is the site that binds oxygen (O2). Thus, each molecule of hemoglobin in the blood carries four molecules of oxygen. (PDB code: 2DHB.) Figure 3–20 Protein assemblies. (A) A protein with just one binding site can (A) form a dimer with another identical protein. (B) Identical proteins with two free assembled different binding sites often form a long helical filament. (C) If the two binding subunits structures sites are disposed appropriately in relation to each other, the protein subunits may form a closed ring instead of a helix. (For an example of A, see


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Cell_Biology_Alberts. Figure 3–18; for an example of B, see Figure 3–21; for examples of C, see Figures 5–14 and 14–31.) site site complementary to another region of the surface of the same molecule (Figure 3–20). An actin filament, for example, is a long helical structure produced from many molecules of the protein actin (Figure 3–21). Actin is a globular protein that is very abundant in eukaryotic cells, where it forms one of the major filament systems of the cytoskeleton (discussed in Chapter 16).


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Cell_Biology_Alberts. We will encounter many helical structures in this book. Why is a helix such a common structure in biology? As we have seen, biological structures are often formed by linking similar subunits into long, repetitive chains. If all the subunits are identical, the neighboring subunits in the chain can often fit together in only one way, adjusting their relative positions to minimize the free energy of the contact between them. As a result, each subunit is positioned in exactly the same way in relation to the next, so that subunit 3 fits onto subunit 2 in the same way that subunit 2 fits onto subunit 1, and so on. Because it is very rare for subunits to join up in a straight line, this arrangement generally results in a helix—a regular structure that resembles a spiral staircase, as illustrated in Figure 3–22. Depending on the twist of the staircase, a helix is said to be either right-handed or left-handed (see Figure 3–22E). Handedness is not affected by turning the helix upside down, but


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Cell_Biology_Alberts. in Figure 3–22. Depending on the twist of the staircase, a helix is said to be either right-handed or left-handed (see Figure 3–22E). Handedness is not affected by turning the helix upside down, but it is reversed if the helix is reflected in the mirror.


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Cell_Biology_Alberts. The observation that helices occur commonly in biological structures holds true whether the subunits are small molecules linked together by covalent bonds (for example, the amino acids in an α helix) or large protein molecules that are linked by noncovalent forces (for example, the actin molecules in actin filaments). This is not surprising. A helix is an unexceptional structure, and it is generated simply by placing many similar subunits next to each other, each in the same strictly repeated relationship to the one before—that is, with a fixed rotation followed by a fixed translation along the helix axis, as in a spiral staircase. Many Protein Molecules Have Elongated, Fibrous Shapes


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Cell_Biology_Alberts. Many Protein Molecules Have Elongated, Fibrous Shapes Enzymes tend to be globular proteins: even though many are large and complicated, with multiple subunits, most have an overall rounded shape. In Figure 3–21, we saw that a globular protein can also associate to form long filaments. But there are also functions that require each individual protein molecule to span a large distance. These proteins generally have a relatively simple, elongated three-dimensional structure and are commonly referred to as fibrous proteins.


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Cell_Biology_Alberts. One large family of intracellular fibrous proteins consists of α-keratin, introduced when we presented the α helix, and its relatives. Keratin filaments are extremely stable and are the main component in long-lived structures such as hair, horn, and nails. An α-keratin molecule is a dimer of two identical subunits, with the long α helices of each subunit forming a coiled-coil (see Figure 3–9). The coiled-coil regions are capped at each end by globular domains containing binding sites. This enables this class of protein to assemble into ropelike intermediate filaments—an important component of the cytoskeleton that creates the cell’s internal structural framework (see Figure 16–67).


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Cell_Biology_Alberts. Fibrous proteins are especially abundant outside the cell, where they are a main component of the gel-like extracellular matrix that helps to bind collections of cells together to form tissues. Cells secrete extracellular matrix proteins into their surroundings, where they often assemble into sheets or long fibrils. Collagen is the most abundant of these proteins in animal tissues. A collagen molecule consists of three long polypeptide chains, each containing the nonpolar amino Figure 3–21 actin filaments. (A) Transmission electron micrographs of negatively stained actin filaments. (B) The helical arrangement of actin molecules in an actin filament. (A, courtesy of Roger Craig.) Figure 3–22 Some properties of a helix.


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Cell_Biology_Alberts. (A–D) A helix forms when a series of subunits bind to each other in a regular way. At the bottom, each of these helices is viewed from directly above the helix and seen to have two (A), three (B), and six (C and D) subunits per helical turn. Note that the helix in (D) has a wider path than that in (C), but the same number of subunits per turn. (E) As discussed in the text, a helix can be either right-handed or left-handed. As a reference, it is useful to remember that standard metal screws, which insert when turned clockwise, are right-handed. Note that a helix retains the same handedness when it is turned upside down. (PDB code: 2DHB.) acid glycine at every third position. This regular structure allows the chains to wind around one another to generate a long regular triple helix (Figure 3–23A). Many collagen molecules then bind to one another side-by-side and end-toend to create long overlapping arrays—thereby generating the extremely tough collagen fibrils that give connective


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Cell_Biology_Alberts. 3–23A). Many collagen molecules then bind to one another side-by-side and end-toend to create long overlapping arrays—thereby generating the extremely tough collagen fibrils that give connective tissues their tensile strength, as described in Chapter 19.


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Cell_Biology_Alberts. Proteins Contain a Surprisingly Large Amount of Intrinsically Disordered Polypeptide Chain It has been well known for a long time that, in complete contrast to collagen, another abundant protein in the extracellular matrix, elastin, is formed as a highly disordered polypeptide. This disorder is essential for elastin’s function. Its relatively loose and unstructured polypeptide chains are covalently cross-linked to short section of 50 nm collagen fbril collagen molecule 300 nm × 1.5 nm 1.5 nm Figure 3–23 Collagen and elastin. (A) Collagen is a triple helix formed by three extended protein chains that wrap around one another (bottom). Many rodlike collagen molecules are cross-linked together in the extracellular space to form unextendable collagen fibrils (top) that have the tensile strength of steel. The striping on the collagen fibril is caused by the regular repeating arrangement of the collagen molecules within the fibril.


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Cell_Biology_Alberts. (B) Elastin polypeptide chains are cross-linked together in the extracellular space to form rubberlike, elastic fibers. Each elastin molecule uncoils into a more extended conformation when the fiber is stretched and recoils spontaneously as soon as the stretching force is relaxed. The cross-linking in the extracellular space mentioned creates covalent linkages between lysine side chains, but the chemistry is different for collagen and elastin. produce a rubberlike, elastic meshwork that can be reversibly pulled from one conformation to another, as illustrated in Figure 3–23B. The elastic fibers that result enable skin and other tissues, such as arteries and lungs, to stretch and recoil without tearing.


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Cell_Biology_Alberts. Intrinsically disordered regions of proteins are frequent in nature, and they have important functions in the interior of cells. As we have already seen, proteins often have loops of polypeptide chain that protrude from the core region of a protein domain to bind other molecules. Some of these loops remain largely unstructured until they bind to a target molecule, adopting a specific folded conformation only when this other molecule is bound. Many proteins were also known to have intrinsically disordered tails at one or the other end of a structured domain (see, for example, the histones in Figure 4–24). But the extent of such disordered structure only became clear when genomes were sequenced. This allowed bioinformatic methods to be used to analyze the amino acid sequences that genes encode, searching for disordered regions based on their unusually low hydrophobicity and relatively high net charge. Combining these results with other data, it is now thought that perhaps a quarter of


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Cell_Biology_Alberts. searching for disordered regions based on their unusually low hydrophobicity and relatively high net charge. Combining these results with other data, it is now thought that perhaps a quarter of all eukaryotic proteins can adopt structures that are mostly disordered, fluctuating rapidly between many different conformations. Many such intrinsically disordered regions contain repeated sequences of amino acids. What do these disordered regions do?


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Cell_Biology_Alberts. Some known functions are illustrated in Figure 3–24. One predominant function is to form specific binding sites for other protein molecules that are of high specificity, but readily altered by protein phosphorylation, protein dephosphorylation, or any of the other covalent modifications that are triggered by cell signaling events (Figure 3–24A and B). We shall see, for example, that the eukaryotic RNA polymerase enzyme that produces mRNAs contains a long, unstructured C-terminal tail that is covalently modified as its RNA synthesis proceeds, thereby attracting specific other proteins to the transcription complex at different times (see Figure 6–22). And this unstructured tail interacts with a different type of low complexity domain when the RNA polymerase is recruited to the specific sites on the DNA where it begins synthesis.


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Cell_Biology_Alberts. As illustrated in Figure 3–24C, an unstructured region can also serve as a “tether” to hold two protein domains in close proximity to facilitate their interaction. For example, it is this tethering function that allows substrates to move between active sites in large multienzyme complexes (see Figure 3–54). A similar tethering function allows large scaffold proteins with multiple protein-binding sites to concentrate sets of interacting proteins, both increasing reaction rates and confining their reaction to a particular site in a cell (see Figure 3–78).


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Cell_Biology_Alberts. Like elastin, other proteins have a function that directly requires that they remain largely unstructured. Thus, large numbers of disordered protein chains in close proximity can create micro-regions of gel-like consistency inside the cell that restrict diffusion. For example, the abundant nucleoporins that coat the inner surface of the nuclear pore complex form a random coil meshwork (Figure 3–24) that is critical for selective nuclear transport (see Figure 12–8).


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Cell_Biology_Alberts. Figure 3–24 Some important functions for intrinsically disordered protein sequences. (A) Unstructured regions of polypeptide chain often form binding sites for other proteins. Although these binding events are of high specificity, they are often of low affinity due to the free-energy cost of folding the normally unfolded partner (and they are thus readily reversible). (B) Unstructured regions can be easily modified covalently to change their binding preferences, and they are therefore frequently involved in cell signaling processes. In this schematic, multiple sites of protein phosphorylation are indicated. (C) Unstructured regions frequently create “tethers” that hold interacting protein domains in close proximity. (D) A dense network of unstructured proteins can form a diffusion barrier, as the nucleoporins do for the nuclear pore.


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Cell_Biology_Alberts. Figure 3–25 Disulfide bonds. Covalent disulfide bonds form between adjacent cysteine side chains. These cross-linkages can join either two parts of the same polypeptide chain or two different polypeptide chains. Since the energy required to break one covalent bond is much larger than the energy required to break even a whole set of noncovalent bonds (see Table 2–1, p. 45), a disulfide bond can have a major stabilizing effect on a protein (Movie 3.7).


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Cell_Biology_Alberts. Many protein molecules are either attached to the outside of a cell’s plasma membrane or secreted as part of the extracellular matrix. All such proteins are directly exposed to extracellular conditions. To help maintain their structures, the polypeptide chains in such proteins are often stabilized by covalent cross-linkages. These linkages can either tie together two amino acids in the same protein, or connect different polypeptide chains in a multisubunit protein. Although many other types exist, the most common cross-linkages in proteins are covalent sulfur– sulfur bonds. These disulfide bonds (also called S–S bonds) form as cells prepare newly synthesized proteins for export. As described in Chapter 12, their formation is catalyzed in the endoplasmic reticulum by an enzyme that links together two pairs of –SH groups of cysteine side chains that are adjacent in the folded protein (Figure 3–25). Disulfide bonds do not change the conformation of a protein but instead act as atomic


Cell_Biology_Alberts

Cell_Biology_Alberts. together two pairs of –SH groups of cysteine side chains that are adjacent in the folded protein (Figure 3–25). Disulfide bonds do not change the conformation of a protein but instead act as atomic staples to reinforce its most favored conformation. For example, lysozyme—an enzyme in tears that dissolves bacterial cell walls—retains its antibacterial activity for a long time because it is stabilized by such cross-linkages.


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Cell_Biology_Alberts. Disulfide bonds generally fail to form in the cytosol, where a high concentration of reducing agents converts S–S bonds back to cysteine –SH groups. Apparently, proteins do not require this type of reinforcement in the relatively mild environment inside the cell. Protein Molecules Often Serve as Subunits for the Assembly of Large Structures The same principles that enable a protein molecule to associate with itself to form rings or a long filament also operate to generate much larger structures formed from a set of different macromolecules, such as enzyme complexes, ribosomes, viruses, and membranes. These large objects are not made as single, giant, covalently linked molecules. Instead they are formed by the noncovalent assembly of many separately manufactured molecules, which serve as the subunits of the final structure. The use of smaller subunits to build larger structures has several advantages: 1.


Cell_Biology_Alberts

Cell_Biology_Alberts. The use of smaller subunits to build larger structures has several advantages: 1. A large structure built from one or a few repeating smaller subunits requires only a small amount of genetic information. 2. Both assembly and disassembly can be readily controlled reversible processes, because the subunits associate through multiple bonds of relatively low energy. 3. Errors in the synthesis of the structure can be more easily avoided, since correction mechanisms can operate during the course of assembly to exclude malformed subunits.


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Cell_Biology_Alberts. 3. Errors in the synthesis of the structure can be more easily avoided, since correction mechanisms can operate during the course of assembly to exclude malformed subunits. Some protein subunits assemble into flat sheets in which the subunits are arranged in hexagonal patterns. Specialized membrane proteins are sometimes arranged this way in lipid bilayers. With a slight change in the geometry of the individual subunits, a hexagonal sheet can be converted into a tube (Figure 3–26) or, with more changes, into a hollow sphere. Protein tubes and spheres that bind specific RNA and DNA molecules in their interior form the coats of viruses.


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Cell_Biology_Alberts. The formation of closed structures, such as rings, tubes, or spheres, provides additional stability because it increases the number of bonds between the protein subunits. Moreover, because such a structure is created by mutually dependent, cooperative interactions between subunits, a relatively small change that affects each subunit individually can cause the structure to assemble or disassemble. These principles are dramatically illustrated in the protein coat or capsid of many simple viruses, which takes the form of a hollow sphere based on an icosahedron (Figure 3–27). Capsids are often made of hundreds of identical protein subunits that enclose and protect the viral nucleic acid (Figure 3–28). The protein in such a capsid must have a particularly adaptable structure: not only must it make several different kinds of contacts to create the sphere, it must also change this arrangement to let the nucleic acid out to initiate viral replication once the virus has entered a cell.


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Cell_Biology_Alberts. Many Structures in Cells Are Capable of Self-Assembly The information for forming many of the complex assemblies of macromolecules in cells must be contained in the subunits themselves, because purified subunits can spontaneously assemble into the final structure under the appropriate conditions. The first large macromolecular aggregate shown to be capable of self-assembly from its component parts was tobacco mosaic virus (TMV ). This virus is a long rod in which a cylinder of protein is arranged around a helical RNA core (Figure 3–29). If the dissociated RNA and protein subunits are mixed together in solution, they recombine to form fully active viral particles. The assembly process is unexpectedly complex and includes the formation of double rings of protein, which serve as intermediates that add to the growing viral coat.


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Cell_Biology_Alberts. Another complex macromolecular aggregate that can reassemble from its component parts is the bacterial ribosome. This structure is composed of about 55 different protein molecules and 3 different rRNA molecules. Incubating a mixture of the individual components under appropriate conditions in a test tube causes them to spontaneously re-form the original structure. Most importantly, such reconstituted ribosomes are able to catalyze protein synthesis. As might be expected, the reassembly of ribosomes follows a specific pathway: after certain proteins have bound to the RNA, this complex is then recognized by other proteins, and so on, until the structure is complete. It is still not clear how some of the more elaborate self-assembly processes are regulated. Many structures in the cell, for example, seem to have a precisely defined length that is many times greater than that of their component macromolecules. How such length determination is achieved is in many cases a mystery. In


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Cell_Biology_Alberts. Figure 3–26 Single protein subunits form protein assemblies that feature multiple protein–protein contacts. Hexagonally packed globular protein subunits are shown here forming either flat sheets or tubes. Generally, such large structures are not considered to be single “molecules.” Instead, like the actin filament described previously, they are viewed as assemblies formed of many different molecules.


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Cell_Biology_Alberts. Figure 3–27 The protein capsid of a virus. The structure of the simian virus SV40 capsid has been determined by x-ray crystallography and, as for the capsids of many other viruses, it is known in atomic detail. (Courtesy of Robert Grant, Stephan Crainic, and James M. Hogle.) capsid protein intact virus monomer particle shown as (90 dimers) the simplest case, a long core protein or other macromolecule provides a scaffold that determines the extent of the final assembly. This is the mechanism that determines the length of the TMV particle, where the RNA chain provides the core. Similarly, a core protein interacting with actin is thought to determine the length of the thin filaments in muscle.


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Cell_Biology_Alberts. Figure 3–28 The structure of a spherical virus. In viruses, many copies of a single protein subunit often pack together to create a spherical shell (a capsid). This capsid encloses the viral genome, composed of either RNA or DNA (see also Figure 3–27). For geometric reasons, no more than 60 identical subunits can pack together in a precisely symmetric way. If slight irregularities are allowed, however, more subunits can be used to produce a larger capsid that retains icosahedral symmetry. The tomato bushy stunt virus (TBSV) shown here, for example, is a spherical virus about 33 nm in diameter formed from 180 identical copies of a 386-amino-acid capsid protein plus an RNA genome of 4500 nucleotides. To construct such a large capsid, the protein must be able to fit into three somewhat different environments. This requires three slightly different conformations, each of which is differently colored in the virus particle shown here. The postulated pathway of assembly is shown; the precise


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Cell_Biology_Alberts. environments. This requires three slightly different conformations, each of which is differently colored in the virus particle shown here. The postulated pathway of assembly is shown; the precise three-dimensional structure has been determined by x-ray diffraction. (Courtesy of Steve Harrison.)


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Cell_Biology_Alberts. Figure 3–29 The structure of tobacco mosaic virus (TMV). (A) An electron micrograph of the viral particle, which consists of a single long RNA molecule enclosed in a cylindrical protein coat composed of identical protein subunits. (B) A model showing part of the structure of TMV. A single-stranded RNA molecule of 6395 nucleotides is packaged in a helical coat constructed from 2130 copies of a coat protein 158 amino acids long. Fully infective viral particles can self-assemble in a test tube from purified RNA and protein molecules. (A, courtesy of Robley Williams; B, courtesy of Richard J. Feldmann.)


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Cell_Biology_Alberts. Figure 3–30 Proteolytic cleavage in insulin assembly. The polypeptide hormone insulin cannot spontaneously re-form efficiently if its disulfide bonds are disrupted. It is synthesized as a larger protein (proinsulin) that is cleaved by a proteolytic enzyme after the protein chain has folded into a specific shape. Excision of part of the proinsulin polypeptide chain removes some of the information needed for the protein to fold spontaneously into its normal conformation. Once insulin has been denatured and its two polypeptide chains have separated, its ability to reassemble is lost. Assembly Factors Often Aid the Formation of Complex Biological Structures


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Cell_Biology_Alberts. Assembly Factors Often Aid the Formation of Complex Biological Structures Not all cellular structures held together by noncovalent bonds self-assemble. A cilium, or a myofibril of a muscle cell, for example, cannot form spontaneously from a solution of its component macromolecules. In these cases, part of the assembly information is provided by special enzymes and other proteins that perform the function of templates, serving as assembly factors that guide construction but take no part in the final assembled structure.


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Cell_Biology_Alberts. Even relatively simple structures may lack some of the ingredients necessary for their own assembly. In the formation of certain bacterial viruses, for example, the head, which is composed of many copies of a single protein subunit, is assembled on a temporary scaffold composed of a second protein that is produced by the virus. Because the second protein is absent from the final viral particle, the head structure cannot spontaneously reassemble once it has been taken apart. Other examples are known in which proteolytic cleavage is an essential and irreversible step in the normal assembly process. This is even the case for some small protein assemblies, including the structural protein collagen and the hormone insulin (Figure 3–30). From these relatively simple examples, it seems certain that the assembly of a structure as complex as a cilium will involve a temporal and spatial ordering that is imparted by numerous other components.


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Cell_Biology_Alberts. A special class of protein structures, utilized for some normal cell functions, can also contribute to human diseases when not controlled. These are self-propagating, stable β-sheet aggregates called amyloid fibrils. These fibrils are built from a series of identical polypeptide chains that become layered one over the other to create a continuous stack of β sheets, with the β strands oriented perpendicular to the fibril axis to form a cross-beta filament (Figure 3–31). Typically, hundreds of monomers will aggregate to form an unbranched fibrous structure that is several micrometers long and 5 to 15 nm in width. A surprisingly large fraction of proteins have the potential to form such structures, because the short segment of the polypeptide chain that forms the spine of the fibril can have a variety of different sequences and follow one of several different paths (Figure 3–32). However, very few proteins will actually form this structure inside cells.


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Cell_Biology_Alberts. In normal humans, the quality control mechanisms governing proteins gradually decline with age, occasionally permitting normal proteins to form pathological aggregates. The protein aggregates may be released from dead cells and accumulate as amyloid in the extracellular matrix. In extreme cases, the accumulation of such amyloid fibrils in the cell interior can kill the cells and damage tissues. Because the brain is composed of a highly organized collection of nerve cells that cannot regenerate, the brain is especially vulnerable to this sort of cumulative damage. Thus, although amyloid fibrils may form in different tissues, and are known to cause pathologies in several sites in the body, the most severe amyloid pathologies are neurodegenerative diseases. For example, the abnormal formation of highly stable amyloid fibrils is thought to play a central causative role in both Alzheimer’s and Parkinson’s diseases.


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Cell_Biology_Alberts. Prion diseases are a special type of these pathologies. They have attained special notoriety because, unlike Parkinson’s or Alzheimer’s, prion diseases can spread from one organism to another, providing that the second organism eats a S S S S S S SH SH SH SH SH SH specifc folding stabilized by disulfde bonds connecting peptide removed, leaving complete two-chain insulin molecule reduction irreversibly separates the two chains


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Cell_Biology_Alberts. Figure 3–31 Detailed structure of the core of an amyloid fibril. Illustrated here is the cross-beta spine of the amyloid fibril that is formed by a peptide of seven amino acids from the protein Sup35, an extensively studied yeast prion. Consisting of the sequence glycine-asparagine-asparagine-glutamine-glutamine-asparaginetyrosine (GNNQQNY), its structure was determined by X-ray crystallography. Although the cross-beta spines of other amyloids are similar, being composed of two long β sheets held together by a “steric zipper,” different detailed structures are observed depending on the short peptide sequence involved. (A) One half of the spine is illustrated. Here, a standard parallel β-sheet structure (see p. 116) is held together by a set of hydrogen bonds between two side chains plus hydrogen bonds between two backbone atoms, as illustrated (oxygen atoms red and nitrogen atoms blue). Note that in this example, the adjacent peptides are exactly in register. Although only five


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Cell_Biology_Alberts. plus hydrogen bonds between two backbone atoms, as illustrated (oxygen atoms red and nitrogen atoms blue). Note that in this example, the adjacent peptides are exactly in register. Although only five layers are shown (each layer depicted as an arrow), the actual structure would extend for many tens of thousands of layers in the plane of the paper. (B) The complete cross-beta spine. A second, identical β-sheet is paired with the first one to form a two-sheet motif that runs the entire length of the fibril. (C) View of the complete spine in (B) from the top. The closely interdigitated side chains form a tight, water-free junction known as a steric zipper. (Courtesy of David Eisenberg and Michael Sawaya, UCLA; based on R. Nelson et al., Nature 435:773–778, 2005. With permission from Macmillan Publishers Ltd.) tissue containing the protein aggregate. A set of closely related diseases—scrapie in sheep, Creutzfeldt–Jakob disease (CJD) in humans, Kuru in humans, and bovine spongiform


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Cell_Biology_Alberts. Publishers Ltd.) tissue containing the protein aggregate. A set of closely related diseases—scrapie in sheep, Creutzfeldt–Jakob disease (CJD) in humans, Kuru in humans, and bovine spongiform encephalopathy (BSE) in cattle—are caused by a misfolded, aggregated form of a particular protein called PrP (for prion protein). PrP is normally located on the outer surface of the plasma membrane, most prominently in neurons, and it has the unfortunate property of forming amyloid fibrils that are “infectious” because they convert normally folded molecules of PrP to the same pathological form (Figure 3–33). This property creates a positive feedback loop that propagates the abnormal form of PrP, called PrP*, and allows the pathological conformation to spread rapidly from cell to cell in the brain, eventually causing death. It can be dangerous to eat the tissues of animals that contain PrP*, as witnessed by the spread of BSE (commonly referred to as “mad cow disease”) from cattle to humans.


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Cell_Biology_Alberts. eventually causing death. It can be dangerous to eat the tissues of animals that contain PrP*, as witnessed by the spread of BSE (commonly referred to as “mad cow disease”) from cattle to humans. Fortunately, in the absence of PrP*, PrP is extraordinarily difficult to convert to its abnormal form.


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Cell_Biology_Alberts. A closely related “protein-only inheritance” has been observed in yeast cells. The ability to study infectious proteins in yeast has clarified another remarkable feature of prions. These protein molecules can form several distinctively different types of amyloid fibrils from the same polypeptide chain. Moreover, each type of aggregate can be infectious, forcing normal protein molecules to adopt the same type of abnormal structure. Thus, several different “strains” of infectious particles can arise from the same polypeptide chain.


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Cell_Biology_Alberts. Figure 3–32 The structure of an amyloid fibril. (A) Schematic diagram of the structure of a amyloid fibril that is formed by the aggregation of a protein. Only the cross-beta spine of an amyloid fibril resembles the structure shown in Figure 3–31. (B) A cut-away view of a structure proposed for the amyloid fibril that can be formed in a test tube by the enzyme ribonuclease A, illustrating how the core of the fibril—formed by a short segment— relates to the rest of the structure. Electron micrograph of amyloid fibrils. (A, from L. Esposito, C. Pedone and L. Vitagliano, Proc. Natl Acad. Sci. USA 103:11533–11538, 2006; B, from S. Sambashivan et al., Nature 437:266–269, 2005; C, courtesy of David Eisenberg.) Figure 3–33 The special protein aggregates that cause prion diseases.


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Cell_Biology_Alberts. Figure 3–33 The special protein aggregates that cause prion diseases. (A) Schematic illustration of the type of conformational change in the PrP protein (prion protein) that produces material for an amyloid fibril. (B) The self-infectious nature of the protein aggregation that is central to prion diseases. PrP is highly unusual because the misfolded version of the protein, called PrP*, induces the normal PrP protein it contacts to change its conformation, as shown.


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Cell_Biology_Alberts. Amyloid fibrils were initially studied because they cause disease. But the same type of structure is now known to be exploited by cells for useful purposes. Eukaryotic cells, for example, store many different peptide and protein hormones that they will secrete in specialized “secretory granules,” which package a high concentration of their cargo in dense cores with a regular structure (see Figure 13–65). We now know that these structured cores consist of amyloid fibrils, which in this case have a structure that causes them to dissolve to release soluble cargo after being secreted by exocytosis to the cell exterior (Figure 3–34A). Many bacteria use the amyloid structure in a very different way, secreting proteins that form long amyloid fibrils projecting from the cell exterior that help to bind bacterial neighbors into biofilms (Figure 3–34B). Because these biofilms help bacteria to survive in adverse environments (including in humans treated with antibiotics), new drugs that


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Cell_Biology_Alberts. help to bind bacterial neighbors into biofilms (Figure 3–34B). Because these biofilms help bacteria to survive in adverse environments (including in humans treated with antibiotics), new drugs that specifically disrupt the fibrous networks formed by bacterial amyloids have promise for treating human infections.


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Cell_Biology_Alberts. Until recently, those amyloids with useful functions were thought to be either confined to the interior of specialized vesicles or expressed on the exterior of cells, as in Figure 3–34. However, new experiments reveal that a large set of low complexity domains can form amyloid fibers that have functional roles in both the cell nucleus and the cell cytoplasm. These domains are normally unstructured and consist of stretches of amino acid sequence that can span hundreds of amino acids, while containing only a small subset of the 20 different amino acids. In contrast to the disease-associated amyloid in Figure 3–33, these newly discovered structures are held together by weaker noncovalent bonds and readily dissociate in response to signals—hence their name reversible amyloids. Many proteins with such domains also contain a different set of domains that bind to specific other protein or RNA molecules. Thus, their controlled aggregation


Cell_Biology_Alberts

Cell_Biology_Alberts. Many proteins with such domains also contain a different set of domains that bind to specific other protein or RNA molecules. Thus, their controlled aggregation Golgi amyloid template subunit cisterna fbril subunit of fbril (A) prion protein can adopt an abnormal, misfolded form normal Prp abnormal prion form protein of PrP protein (Prp*) (B) misfolded protein can induce formation of protein aggregates the conversion of more PrP to misfolded form creates a stable amyloid fbril protein aggregate in form of amyloid fbril


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Cell_Biology_Alberts. Figure 3–34 Two normal functions for amyloid fibrils. (A) In eukaryotic cells, protein cargo can be packed very densely in secretory vesicles and stored until signals cause a release of this cargo by exocytosis. For example, proteins and peptide hormones of the endocrine system, such as glucagon and calcitonin, are efficiently stored as short amyloid fibrils, which dissociate when they reach the cell exterior. (B) Bacteria produce amyloid fibrils on their surface by secreting the precursor proteins; these fibrils then create biofilms that link together, and help to protect, large numbers of individual bacteria. soluble protein with green fuorescent tag hnRNPA2 t/2 = 10.1 minSOLUBLE PROTEIN REPLACED BY BUFFER hnRNPA1 t/2 = 3.6 min 0.5 1 2 3 5 101520304560dissociation of green protein from time after washinggel is measured by fuorescence (A) microscope as a function of time (B)


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Cell_Biology_Alberts. Figure 3–35 Measuring the association between “reversible amyloids.” (A) Experimental setup. The fiber-forming domains from proteins that contain a low-complexity domain were produced in large quantities by cloning the DNA sequence that encodes them into an E. coli plasmid so as to allow overproduction of that domain (see p. 483). After these protein domains were purified by affinity chromatography, a tiny droplet of concentrated solution of one of the domains (here the FUS low-complexity domain) was deposited onto a microscope dish and allowed to gel. The gel was then soaked in a dilute solution of a fluorescently labeled low-complexity domain from the same or a different protein, making the gel fluorescent. After replacing the dilute protein solution with buffer, the relative strength of binding of the various domains to each other could then be measured by the decay of fluorescence, as indicated. (B) Results. The low-complexity domain from the FUS protein binds more tightly to


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Cell_Biology_Alberts. of binding of the various domains to each other could then be measured by the decay of fluorescence, as indicated. (B) Results. The low-complexity domain from the FUS protein binds more tightly to itself than it does to the low-complexity domains from the proteins hnRNPA1 or hnRNPA2. A separate experiment reveals that these three different RNA binding proteins associate by forming mixed amyloid fibrils. (Adapted from M.Kato et al., Cell 149: 753-767, 2012).


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Cell_Biology_Alberts. in the cell can form a hydrogel that pulls these and other molecules into punctate structures called intracellular bodies, or granules. Specific mRNAs can be sequestered in such granules, where they are stored until made available by a controlled disassembly of the core amyloid structure that holds them together.


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Cell_Biology_Alberts. Consider the FUS protein, an essential nuclear protein with roles in the transcription, processing, and transport of specific mRNA molecules. Over 80 percent of its C-terminal domain of two hundred amino acids is composed of only four amino acids: glycine, serine, glutamine, and tyrosine. This low complexity domain is attached to several other domains that bind to RNA molecules. At high enough concentrations in a test tube, it forms a hydrogel that will associate with either itself or with the low complexity domains from other proteins. As illustrated by the experiment in Figure 3–35, although different low complexity domains bind to each other, homotypic interactions appear to be of greatest affinity (thus, the FUS low complexity domain binds most tightly to itself). Further experiments reveal that that both the homotypic and the heterotypic bindings are mediated through a β-sheet core structure forming amyloid fibrils, and that these structures bind to other types of repeat


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Cell_Biology_Alberts. reveal that that both the homotypic and the heterotypic bindings are mediated through a β-sheet core structure forming amyloid fibrils, and that these structures bind to other types of repeat sequences in the manner indicated in Figure 3–36. Many of these interactions appear to be controlled by the phosphorylation of serine side chains in the one or both of the interacting partners. However, a great deal remains to be learned concerning these newly discovered structures and the varied roles that they play in the cell biology of eukaryotic cells.


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Cell_Biology_Alberts. protein with low-complexity domain weak cross-beta spine binding site for other proteins with repeated sequences or for RNA molecules Figure 3–36 One type of complex that is formed by reversible amyloids. The structure shown is based on the observed interaction of RNA polymerase with a low-complexity domain of a protein that regulates DNA transcription. (Adapted from I. Kwon et al., Cell 155:1049–1060, 2013.) A protein molecule’s amino acid sequence determines its three-dimensional conformation. Noncovalent interactions between different parts of the polypeptide chain stabilize its folded structure. The amino acids with hydrophobic side chains tend to cluster in the interior of the molecule, and local hydrogen-bond interactions between neighboring peptide bonds give rise to α helices and β sheets.


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Cell_Biology_Alberts. Regions of amino acid sequence known as domains are the modular units from which many proteins are constructed. Such domains generally contain 40–350 amino acids, often folded into a globular shape. Small proteins typically consist of only a single domain, while large proteins are formed from multiple domains linked together by various lengths of polypeptide chain, some of which can be relatively disordered. As proteins have evolved, domains have been modified and combined with other domains to construct large numbers of new proteins.


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Cell_Biology_Alberts. Proteins are brought together into larger structures by the same noncovalent forces that determine protein folding. Proteins with binding sites for their own surface can assemble into dimers, closed rings, spherical shells, or helical polymers. The amyloid fibril is a long unbranched structure assembled through a repeating aggregate of β sheets. Although some mixtures of proteins and nucleic acids can assemble spontaneously into complex structures in a test tube, not all structures in the cell are capable of spontaneous reassembly after they have been dissociated into their component parts, because many biological assembly processes involve assembly factors that are not present in the final structure.


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Cell_Biology_Alberts. We have seen that each type of protein consists of a precise sequence of amino acids that allows it to fold up into a particular three-dimensional shape, or conformation. But proteins are not rigid lumps of material. They often have precisely engineered moving parts whose mechanical actions are coupled to chemical events. It is this coupling of chemistry and movement that gives proteins the extraordinary capabilities that underlie the dynamic processes in living cells. In this section, we explain how proteins bind to other selected molecules and how a protein’s activity depends on such binding. We show that the ability to bind to other molecules enables proteins to act as catalysts, signal receptors, switches, motors, or tiny pumps. The examples we discuss in this chapter by no means exhaust the vast functional repertoire of proteins. You will encounter the specialized functions of many other proteins elsewhere in this book, based on similar principles.


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Cell_Biology_Alberts. All Proteins Bind to Other Molecules A protein molecule’s physical interaction with other molecules determines its biological properties. Thus, antibodies attach to viruses or bacteria to mark them for destruction, the enzyme hexokinase binds glucose and ATP so as to catalyze a reaction between them, actin molecules bind to each other to assemble into actin filaments, and so on. Indeed, all proteins stick, or bind, to other molecules. In some cases, this binding is very tight; in others it is weak and short-lived. But the binding always shows great specificity, in the sense that each protein molecule can usually bind just one or a few molecules out of the many thousands of different types it encounters. The substance that is bound by the protein—whether it is an ion, a small molecule, or a macromolecule such as another protein—is referred to as a ligand for that protein (from the Latin word ligare, meaning “to bind”).


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Cell_Biology_Alberts. The ability of a protein to bind selectively and with high affinity to a ligand depends on the formation of a set of weak noncovalent bonds—hydrogen bonds, electrostatic attractions, and van der Waals attractions—plus favorable hydrophobic interactions (see Panel 2–3, pp. 94–95). Because each individual bond is weak, effective binding occurs only when many of these bonds form simultaneously. Such binding is possible only if the surface contours of the ligand molecule fit very closely to the protein, matching it like a hand in a glove (Figure 3–37).


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Cell_Biology_Alberts. The region of a protein that associates with a ligand, known as the ligand’s binding site, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids. These amino acids can belong to different portions of the polypeptide chain that are brought together when the protein folds (Figure 3–38). Separate regions of the protein surface generally provide binding sites for different ligands, allowing the protein’s activity to be regulated, as we shall see later. And other parts of the protein act as a handle to position the protein in the cell—an example is the SH2 domain discussed previously, which often moves a protein containing it to particular intracellular sites in response to signals.


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Cell_Biology_Alberts. Although the atoms buried in the interior of the protein have no direct contact with the ligand, they form the framework that gives the surface its contours and its chemical and mechanical properties. Even small changes to the amino acids in the interior of a protein molecule can change its three-dimensional shape enough to destroy a binding site on the surface. The Surface Conformation of a Protein Determines Its Chemistry The impressive chemical capabilities of proteins often require that the chemical groups on their surface interact in ways that enhance the chemical reactivity of one or more amino acid side chains. These interactions fall into two main categories. First, the interaction of neighboring parts of the polypeptide chain may restrict the access of water molecules to that protein’s ligand-binding sites. Because water molecules readily form hydrogen bonds that can compete with ligands for sites


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Cell_Biology_Alberts. Figure 3–37 The selective binding of a protein to another molecule. Many weak bonds are needed to enable a protein to bind tightly to a second molecule, or ligand. A ligand must therefore fit precisely into a protein’s binding site, like a hand into a glove, so that a large number of noncovalent bonds form between the protein and the ligand. (A) Schematic; (B) space-filling model. (PDB code: 1G6N.) Figure 3–38 The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can form noncovalent bonds only with certain ligands. (B) A close-up of an actual binding site, showing the hydrogen bonds and electrostatic interactions formed between a protein and its ligand. In this example, a molecule of cyclic AMP is the bound ligand.


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Cell_Biology_Alberts. on the protein surface, a ligand will form tighter hydrogen bonds (and electrostatic interactions) with a protein if water molecules are kept away. It might be hard to imagine a mechanism that would exclude a molecule as small as water from a protein surface without affecting the access of the ligand itself. However, because of the strong tendency of water molecules to form water–water hydrogen bonds, water molecules exist in a large hydrogen-bonded network (see Panel 2–2, pp. 92–93). In effect, a protein can keep a ligand-binding site dry, increasing that site's reactivity, because it is energetically unfavorable for individual water molecules to break away from this network—as they must do to reach into a crevice on a protein’s surface.


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Cell_Biology_Alberts. Second, the clustering of neighboring polar amino acid side chains can alter their reactivity. If protein folding forces together a number of negatively charged side chains against their mutual repulsion, for example, the affinity of the site for a positively charged ion is greatly increased. In addition, when amino acid side chains interact with one another through hydrogen bonds, normally unreactive groups (such as the –CH2OH on the serine shown in Figure 3–39) can become reactive, enabling them to be used to make or break selected covalent bonds. The surface of each protein molecule therefore has a unique chemical reactivity that depends not only on which amino acid side chains are exposed, but also on their exact orientation relative to one another. For this reason, two slightly different conformations of the same protein molecule can differ greatly in their chemistry.


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Cell_Biology_Alberts. As we have described previously, genome sequences allow us to group many of the domains in proteins into families that show clear evidence of their evolution from a common ancestor. The three-dimensional structures of members of the same domain family are remarkably similar. For example, even when the amino acid sequence identity falls to 25%, the backbone atoms in a domain can follow a common protein fold within 0.2 nanometers (2 Å).


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Cell_Biology_Alberts. We can use a method called evolutionary tracing to identify those sites in a protein domain that are the most crucial to the domain’s function. Those sites that bind to other molecules are the most likely to be maintained, unchanged as organisms evolve. Thus, in this method, those amino acids that are unchanged, or nearly unchanged, in all of the known protein family members are mapped onto a model of the three-dimensional structure of one family member. When this is done, the most invariant positions often form one or more clusters on the protein surface, as illustrated in Figure 3–40A for the SH2 domain described previously (see Figure 3–6). These clusters generally correspond to ligand-binding sites.


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Cell_Biology_Alberts. The SH2 domain functions to link two proteins together. It binds the protein containing it to a second protein that contains a phosphorylated tyrosine side chain in a specific amino acid sequence context, as shown in Figure 3–40B. The amino acids located at the binding site for the phosphorylated polypeptide have been the slowest to change during the long evolutionary process that produced Figure 3–39 an unusually reactive amino acid at the active site of an enzyme. This example is the “catalytic triad” Asp-His-Ser found in chymotrypsin, elastase, and other serine proteases (see Figure 3–12). The aspartic acid side chain (Asp) induces the histidine (His) to remove the proton from a particular serine (Ser). This activates the serine and enables it to form a covalent bond with an enzyme substrate, hydrolyzing a peptide bond. The many convolutions of the polypeptide chain are omitted here.


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Cell_Biology_Alberts. the large SH2 family of peptide recognition domains. Mutation is a random process; survival is not. Thus, natural selection (random mutation followed by nonrandom survival) produces the sequence conservation by preferentially eliminating organisms whose SH2 domains become altered in a way that inactivates the SH2 binding site, destroying SH2 function. Genome sequencing has revealed huge numbers of proteins whose functions are unknown. Once a three-dimensional structure has been determined for one member of a protein family, evolutionary tracing allows biologists to determine binding sites for the members of that family, providing a useful start in deciphering protein function. Proteins Bind to Other Proteins Through Several Types of Interfaces


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Cell_Biology_Alberts. Proteins Bind to Other Proteins Through Several Types of Interfaces Proteins can bind to other proteins in multiple ways. In many cases, a portion of the surface of one protein contacts an extended loop of polypeptide chain (a “string”) on a second protein (Figure 3–41A). Such a surface–string interaction, for example, allows the SH2 domain to recognize a phosphorylated polypeptide loop on a second protein, as just described, and it also enables a protein kinase to recognize the proteins that it will phosphorylate (see below). A second type of protein–protein interface forms when two α helices, one from each protein, pair together to form a coiled-coil (Figure 3–41B). This type of protein interface is found in several families of gene regulatory proteins, as discussed in Chapter 7.


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Cell_Biology_Alberts. The most common way for proteins to interact, however, is by the precise matching of one rigid surface with that of another (Figure 3–41C). Such interactions can be very tight, since a large number of weak bonds can form between two surfaces that match well. For the same reason, such surface–surface interactions can be extremely specific, enabling a protein to select just one partner from the many thousands of different proteins found in a cell. Figure 3–40 The evolutionary trace method applied to the SH2 domain.


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Cell_Biology_Alberts. Figure 3–40 The evolutionary trace method applied to the SH2 domain. Front and back views of a space-filling model of the SH2 domain, with evolutionarily conserved amino acids on the protein surface colored yellow, and those more toward the protein interior colored red. (B) The structure of one specific SH2 domain with its bound polypeptide. Here, those amino acids located within 0.4 nm of the bound ligand are colored blue. The two key amino acids of the ligand are yellow, and the others are purple. Note the high degree of correspondence between and (B). (Adapted from O. Lichtarge, H.R. Bourne and F.E. Cohen, J. Mol. Biol. 257:342–358, 1996. With permission from Elsevier; PDB codes: 1SPR, 1SPS.)


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Cell_Biology_Alberts. H.R. Bourne and F.E. Cohen, J. Mol. Biol. 257:342–358, 1996. With permission from Elsevier; PDB codes: 1SPR, 1SPS.) Figure 3–41 Three ways in which two proteins can bind to each other. Only the interacting parts of the two proteins are shown. (A) A rigid surface on one protein can bind to an extended loop of polypeptide chain (a “string”) on a second protein. (B) Two α helices can bind together to form a coiled-coil. (C) Two complementary rigid surfaces often link two proteins together. Binding interactions can also involve the pairing of β strands (see, for example, Figure 3–18).


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Cell_Biology_Alberts. (C) Two complementary rigid surfaces often link two proteins together. Binding interactions can also involve the pairing of β strands (see, for example, Figure 3–18). hypervariable loops variable domain of light chain (VL) (B) constant domain of light chain (CL) disulfde bond COOH NH2 S S S S SS SSSSSSSSVL CL VH CH1CH1 CH2 CH3 Figure 3–42 an antibody molecule. A typical antibody molecule is Y-shaped and has two identical binding sites for its antigen, one on each arm of the Y. As explained in Chapter 24, the protein is composed of four polypeptide chains (two identical heavy chains and two identical and smaller light chains) held together by disulfide bonds. Each chain is made All proteins must bind to particular ligands to carry out their various functions. The antibody family is notable for its capacity for tight, highly selective binding (discussed in detail in Chapter 24).


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Cell_Biology_Alberts. Antibodies, or immunoglobulins, are proteins produced by the immune system in response to foreign molecules, such as those on the surface of an invading microorganism. Each antibody binds tightly to a particular target molecule, thereby either inactivating the target molecule directly or marking it for destruction. An antibody recognizes its target (called an antigen) with remarkable specificity. Because there are potentially billions of different antigens that humans might encounter, we have to be able to produce billions of different antibodies.


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Cell_Biology_Alberts. Antibodies are Y-shaped molecules with two identical binding sites that are complementary to a small portion of the surface of the antigen molecule. A detailed examination of the antigen-binding sites of antibodies reveals that they are formed from several loops of polypeptide chain that protrude from the ends of a pair of closely juxtaposed protein domains (Figure 3–42). Different antibodies generate an enormous diversity of antigen-binding sites by changing only the length and amino acid sequence of these loops, without altering the basic protein structure. Loops of this kind are ideal for grasping other molecules. They allow a large number of chemical groups to surround a ligand so that the protein can link to it with many weak bonds. For this reason, loops often form the ligand-binding sites in proteins. The Equilibrium Constant Measures Binding Strength


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Cell_Biology_Alberts. The Equilibrium Constant Measures Binding Strength Molecules in the cell encounter each other very frequently because of their continual random thermal movements. Colliding molecules with poorly matching surfaces form few noncovalent bonds with one another, and the two molecules dissociate as rapidly as they come together. At the other extreme, when many noncovalent bonds form between two colliding molecules, the association can persist for a very long time (Figure 3–43). Strong interactions occur in cells whenever a biological function requires that molecules remain associated for a long time—for example, when a group of RNA and protein molecules come together to make a subcellular structure such as a ribosome.


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Cell_Biology_Alberts. up of several different immunoglobulin domains, here shaded either blue or gray. The antigen-binding site is formed where a heavy-chain variable domain (VH) and a light-chain variable domain (VL) come close together. These are the domains that differ most in their sequence and structure in different antibodies. At the end of each of the two arms of the antibody molecule, these two domains form loops that bind to the antigen (see Movie 24.5). the surfaces of molecules A and B, and A and C, are a poor match and are capable of forming only a few weak bonds; thermal motion rapidly breaks them apart molecule A randomly encounters other molecules (B, C, and D) the surfaces of molecules A and D match well and therefore can form enough weak bonds to withstand thermal jolting; they therefore


Cell_Biology_Alberts

Cell_Biology_Alberts. We can measure the strength with which any two molecules bind to each other. As an example, consider a population of identical antibody molecules that suddenly encounters a population of ligands diffusing in the fluid surrounding them. At frequent intervals, one of the ligand molecules will bump into the binding site of an antibody and form an antibody–ligand complex. The population of antibody–ligand complexes will therefore increase, but not without limit: over time, a second process, in which individual complexes break apart because of thermally induced motion, will become increasingly important. Eventually, any population of antibody molecules and ligands will reach a steady state, or equilibrium, in which the number of binding (association) events per second is precisely equal to the number of “unbinding” (dissociation) events (see Figure 2–30).


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Cell_Biology_Alberts. From the concentrations of the ligand, antibody, and antibody–ligand complex at equilibrium, we can calculate a convenient measure of the strength of bind-ing—the equilibrium constant (K)—(Figure 3–44A). This constant was described in detail in Chapter 2, where its connection to free energy differences was derived (see p. 62). The equilibrium constant for a reaction in which two molecules (A and B) bind to each other to form a complex (AB) has units of liters/mole, and half of the binding sites will be occupied by ligand when that ligand’s concentration (in moles/liter) reaches a value that is equal to 1/K. This equilibrium constant is larger the greater the binding strength, and it is a direct measure of the free-energy difference between the bound and free states (Figure 3–44B). Even a change stay bound to each other Figure 3–43 How noncovalent bonds mediate interactions between macromolecules (see Movie 2.1).


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Cell_Biology_Alberts. Figure 3–44 Relating standard free-energy difference (ΔG°) to the equilibrium constant (K). (A) The equilibrium between molecules A and B and the complex AB is maintained by a balance between the two opposing reactions shown in panels 1 and 2. Molecules A and B must collide if they are to react, and the association rate is therefore proportional to the product of their individual concentrations [A] × [B]. (Square brackets indicate concentration.) As shown in panel 3, the ratio of the rate constants for the association and the dissociation reactions is equal to the equilibrium constant (K) for the reaction (see also p. 63). (B) The equilibrium constant in panel 3 is that for the reaction A + B ↔ AB, and the larger its value, the stronger the binding between A and B. Note that for every 5.91 kJ/mole decrease in standard free energy, the equilibrium constant increases by a factor of 10 at 37°C.


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Cell_Biology_Alberts. The equilibrium constant here has units of liters/mole; for simple binding interactions it is also called the affinity constant or association constant, denoted Ka. The reciprocal of Ka is called the dissociation 1 2 3 dissociation AB A + B dissociation rate = dissociation rate constant ×concentration of AB dissociation rate = koff [AB] association ABA + B association rate = association rate constant ×concentration of A association rate = kon [A] [B] ×concentration of B AT EQUILIBRIUM: association rate = dissociation rate kon [A] [B] = koff [AB] [A][B] koff [AB] kon = = K = equilibrium constant equilibrium constant [A][B] [AB] = K(liters/mole) 1 10 102 103 104 105 106 107 108 109 1010 standard free-energy difference of AB minus free energy of A + B (kJ/mole) 0 –5.9 –11.9 –17.8 –23.7 –29.7 –35.6 –41.5 –47.4 –53.4 –59.4 The relationship between standard free-energy differences (°G°) and equilibrium constants (37°C) (A) (B) constant, Kd (in units of moles/liter).


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Cell_Biology_Alberts. of a few noncovalent bonds can have a striking effect on a binding interaction, as shown by the example in Figure 3–45. (Note that the equilibrium constant, as defined here, is also known as the association or affinity constant, Ka.) We have used the case of an antibody binding to its ligand to illustrate the effect of binding strength on the equilibrium state, but the same principles apply to any molecule and its ligand. Many proteins are enzymes, which, as we now discuss, first bind to their ligands and then catalyze the breakage or formation of covalent bonds in these molecules.


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Cell_Biology_Alberts. Many proteins can perform their function simply by binding to another molecule. An actin molecule, for example, need only associate with other actin molecules to form a filament. There are other proteins, however, for which ligand binding is only a necessary first step in their function. This is the case for the large and very important class of proteins called enzymes. As described in Chapter 2, enzymes are remarkable molecules that cause the chemical transformations that make and break covalent bonds in cells. They bind to one or more ligands, called substrates, and convert them into one or more chemically modified products, doing this over and over again with amazing rapidity. Enzymes speed up reactions, often by a factor of a million or more, without themselves being changed—that is, they act as catalysts that permit cells to make or break covalent bonds in a controlled way. It is the catalysis of organized sets of chemical reactions by enzymes that creates and maintains the cell,


Cell_Biology_Alberts

Cell_Biology_Alberts. act as catalysts that permit cells to make or break covalent bonds in a controlled way. It is the catalysis of organized sets of chemical reactions by enzymes that creates and maintains the cell, making life possible.


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Cell_Biology_Alberts. We can group enzymes into functional classes that perform similar chemical reactions (Table 3–1). Each type of enzyme within such a class is highly specific,


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Cell_Biology_Alberts. Figure 3–45 Small changes in the number of weak bonds can have drastic effects on a binding interaction. This example illustrates the dramatic effect of the presence or absence of a few weak noncovalent bonds in a biological context. Consider 1000 molecules of A and 1000 molecules of B in a eukaryotic cell. The concentration of both will be about 10–9 M. If the equilibrium constant (K ) for A + B AB is 1010, then one can calculate that at equilibrium there will be If the equilibrium constant is a little weaker at 108, which represents a loss of 11.9 kilojoule/mole of binding energy from the example above, or 2–3 fewer hydrogen bonds, then there will be 270 A molecules 270 B molecules 730 AB molecules 915 A molecules 915 B molecules 85 AB molecules 0.5Vmax catalyzing only a single type of reaction. Thus, hexokinase adds a phosphate group to D-glucose but ignores its optical isomer L-glucose; the blood-clotting enzyme thrombin cuts one type of blood protein between a particular arginine


Cell_Biology_Alberts

Cell_Biology_Alberts. Thus, hexokinase adds a phosphate group to D-glucose but ignores its optical isomer L-glucose; the blood-clotting enzyme thrombin cuts one type of blood protein between a particular arginine and its adjacent glycine and nowhere else, and so on. As discussed in detail in Chapter 2, enzymes work in teams, with the product of one enzyme becoming the substrate for the next. The result is an elaborate network of metabolic pathways that provides the cell with energy and generates the many large and small molecules that the cell needs (see Figure 2–63).


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Cell_Biology_Alberts. Substrate Binding Is the First Step in Enzyme Catalysis


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Cell_Biology_Alberts. For a protein that catalyzes a chemical reaction (an enzyme), the binding of each substrate molecule to the protein is an essential prelude. In the simplest case, if we denote the enzyme by E, the substrate by S, and the product by P, the basic reaction path is E + S → ES →EP → E + P. There is a limit to the amount of substrate that a single enzyme molecule can process in a given time. Although an increase in the concentration of substrate increases the rate at which product is formed, this rate eventually reaches a maximum value (Figure 3–46). At that point the enzyme molecule is saturated with substrate, and the rate of reaction (Vmax) depends only on how rapidly the enzyme can process the substrate molecule. This maximum rate divided by the enzyme concentration is called the turnover number. Turnover numbers are often about 1000 substrate molecules processed per second per enzyme molecule, although turnover numbers between 1 and 10,000 are known.


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Cell_Biology_Alberts. The other kinetic parameter frequently used to characterize an enzyme is its Km, the concentration of substrate that allows the reaction to proceed at one-half its maximum rate (0.5 Vmax) (see Figure 3–46). A low Km value means that the enzyme reaches its maximum catalytic rate at a low concentration of substrate and generally indicates that the enzyme binds to its substrate very tightly, whereas a high Km value corresponds to weak binding. The methods used to characterize enzymes in this way are explained in Panel 3–2 (pp. 142–143).


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Cell_Biology_Alberts. Enzymes achieve extremely high rates of chemical reaction—rates that are far higher than for any synthetic catalysts. There are several reasons for this efficiency. First, when two molecules need to react, the enzyme greatly increases the local concentration of both of these substrate molecules at the catalytic site, holding them in the correct orientation for the reaction that is to follow. More importantly, however, some of the binding energy contributes directly to the catalysis. Substrate molecules must pass through a series of intermediate states of altered geometry and electron distribution before they form the ultimate products of the reaction. The free energy required to attain the most unstable intermediate state, called the transition state, is known as the activation energy for the reaction, and it is the major determinant of the reaction rate. Enzymes have a much higher affinity for the transition state of the substrate than they have for the stable form.


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Cell_Biology_Alberts. rate of reaction Figure 3–46 enzyme kinetics. The rate of an enzyme reaction (V) increases as the substrate concentration increases until a maximum value (Vmax) is reached. At this point all substrate-binding sites on the enzyme molecules are fully occupied, and the rate of reaction is limited by the rate of the catalytic process on the enzyme surface. For most enzymes, the concentration of substrate at which the reaction rate is half-maximal (Km) is a measure of how tightly the substrate is bound, with a large value of Km corresponding to weak binding.


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Cell_Biology_Alberts. WHY ANALYZE THE KINETICS OF ENZYMES? Enzymes are the most selective and powerful catalysts known. An understanding of their detailed mechanisms provides a critical tool for the discovery of new drugs, for the large-scale industrial synthesis of useful chemicals, and for appreciating the chemistry of cells and organisms. A detailed study of the rates of the chemical reactions that are catalyzed by a purifed enzyme—more specifcally how these rates change with changes in conditions such as the concentrations of substrates, products, inhibitors, and regulatory ligands—allows biochemists to fgure out exactly how each enzyme works. For example, this is the way that the ATP-producing reactions of glycolysis, shown previously in Figure 2–48, were deciphered—allowing us to appreciate the rationale for this critical enzymatic pathway. In this Panel, we introduce the important feld of enzyme kinetics, which has been indispensable for deriving much of the detailed knowledge that we now have about


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Cell_Biology_Alberts. for this critical enzymatic pathway. In this Panel, we introduce the important feld of enzyme kinetics, which has been indispensable for deriving much of the detailed knowledge that we now have about cell chemistry. 142 Panel 3–2: Some of the Methods Used to Study enzymes


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Cell_Biology_Alberts. STEADY-STATE ENZYME KINETICS Many enzymes have only one substrate, which they bind and then process to produce products according to the scheme outlined in Figure 3–50A. In this case, the reaction is written as Here we have assumed that the reverse reaction, in which E + P recombine to form EP and then ES, occurs so rarely that we can ignore it. In this case, EP need not be represented, and we can express the rate of the reaction—known as its velocity, V, as where [ES] is the concentration of the enzyme–substrate complex, and kcat is the turnover number, a rate constant that has a value equal to the number of substrate molecules processed per enzyme molecule each second. But how does the value of [ES] relate to the concentrations that we know directly, which are the total concentration of the enzyme, [Eo], and the concentration of the substrate, [S]? When enzyme and substrate are frst mixed, the concentration [ES] will rise rapidly from zero to a so-called steady-state level, as


Cell_Biology_Alberts

Cell_Biology_Alberts. of the enzyme, [Eo], and the concentration of the substrate, [S]? When enzyme and substrate are frst mixed, the concentration [ES] will rise rapidly from zero to a so-called steady-state level, as illustrated below. E + S E + P ES k1 k–1 kcat V = kcat [ES]


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Cell_Biology_Alberts. At this steady state, [ES] is nearly constant, so that or, since the concentration of the free enzyme, [E], is equal to [Eo] – [ES], Rearranging, and defning the constant Km as we get or, remembering that V = kcat [ES], we obtain the famous Michaelis–Menten equation As [S] is increased to higher and higher levels, essentially all of the enzyme will be bound to substrate at steady state; at this point, a maximum rate of reaction, Vmax , will be reached where V = Vmax = kcat [Eo]. Thus, it is convenient to rewrite the Michaelis–Menten equation as rate of ES formation k1 [E][S] rate of ES breakdown k–1 [ES] + kcat [ES] = k1 k–1 + kcat [ES] = [E][S] = [Eo] – [ES] [S] k1 k–1 + kcat k1 k–1 + kcat Km + [S] kcat [Eo][S] [ES] = [Eo][S] Km + [S] V = Km + [S] Vmax [S]V = THE DOUBLE-RECIPROCAL PLOT A typical plot of V versus [S] for an enzyme that follows Michaelis–Menten kinetics is shown below. From this plot, neither the value of Vmax nor of Km is immediately clear.


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Cell_Biology_Alberts. THE SIGNIFICANCE OF Km, kcat, and kcat /Km As described in the text, Km is an approximate measure of substrate affnity for the enzyme: it is numerically equal to the concentration of [S] at V = 0.5 Vmax. In general, a lower value of Km means tighter substrate binding. In fact, for those cases where kcat is much smaller than k–1, the Km will be equal to Kd, the dissociation constant for substrate binding to the enzyme (Kd = 1/Ka; see Figure 3–44). We have seen that k cat is the turnover number for the enzyme. At very low substrate concentrations, where 20 0 0 2 4 6 8 40 60 80 [S] mmole/liter To obtain Vmax and Km from such data, a double-reciprocal [S] << Km, most of the enzyme is free. Thus we can think of [E] = [Eo], so that the Michaelis–Menten equation becomes V = kcat/Km [E][S]. Thus, the ratio kcat/Km is equivalent to the rate constant for the reaction between free enzyme and free substrate. A comparison of kcat/Km for the same enzyme with different substrates, or for two enzymes


Cell_Biology_Alberts

Cell_Biology_Alberts. the ratio kcat/Km is equivalent to the rate constant for the reaction between free enzyme and free substrate. A comparison of kcat/Km for the same enzyme with different substrates, or for two enzymes with their different substrates, is widely used as a measure of enzyme effectiveness. For simplicity, in this Panel we have discussed enzymes that have only one substrate, such as the lysozyme enzyme described in the text (see p. 144). Most enzymes have two substrates, one of which is often an active carrier


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Cell_Biology_Alberts. SOME ENZYMES ARE DIFFUSION LIMITED The values of kcat, Km, and kcat /Km for some selected molecule—such as NADH or ATP. A similar, but more complex, analysis is used to determine the kinetics of such enzymes—allowing the order of substrate binding and the presence of covalent intermediates along the pathway to be revealed.


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Cell_Biology_Alberts. plot is often used, in which the Michaelis–Menten equation has merely been rearranged, so that 1/V can be plotted versus 1/ [S]. 1/V= + 1/ Vmax Km Vmax [S] 1 123468 1 Vmax 0.01 0.02 0.03 0.04 slope = KM / Vmax 1/V (second/µmole) enzymes are given below: Because an enzyme and its substrate must collide before they can react, kcat /Km has a maximum possible value that is limited by collision rates. If every collision forms an enzyme–substrate complex, one can calculate from diffusion theory that kcat /Km will be between 108 and 109 sec–1M–1, in the case where all subsequent steps proceed immediately. – 0.25 0 0.25 0.5 0.75 1.0– 0.5 fumarase fumarate 8x102 5x10–6 1.6x108 catalase H2O2 4x107 1 4x107 acetylcholinesterase acetylcholine 1.4x104 9x10–5 1.6x108 enzyme substrate kcat (sec–1) kcat/Km (sec–1M–1) Km (M)


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Cell_Biology_Alberts. Thus, it is claimed that enzymes like acetylcholinesterase and1 liter/mmole fumarase are “perfect enzymes,” each enzyme having evolved to the point where nearly every collision with its substrate converts the substrate to a product. [S] Km Figure 3–47 enzymatic acceleration of chemical reactions by decreasing the activation energy. There is a single transition state in this example. However, often both the uncatalyzed reaction (A) and the enzyme-catalyzed reaction (B) go through a series of transition states. In that case, it is the transition state with the highest energy (ST and EST) that determines the activation energy and limits the rate of the reaction. (S = substrate; P = product of the reaction; ES = enzyme–substrate complex; EP = enzyme– product complex.) Because this tight binding greatly lowers the energy of the transition state, the enzyme greatly accelerates a particular reaction by lowering the activation energy that is required (Figure 3–47).


Cell_Biology_Alberts

Cell_Biology_Alberts. Because this tight binding greatly lowers the energy of the transition state, the enzyme greatly accelerates a particular reaction by lowering the activation energy that is required (Figure 3–47). Figure 3–48 compares the spontaneous reaction rates and the corresponding enzyme-catalyzed rates for five enzymes. Rate accelerations range from 109 to 1023. Enzymes not only bind tightly to a transition state, they also contain precisely positioned atoms that alter the electron distributions in the atoms that participate directly in the making and breaking of covalent bonds. Peptide bonds, for example, can be hydrolyzed in the absence of an enzyme by exposing a polypeptide to either a strong acid or a strong base. Enzymes are unique, however, in being able to use acid and base catalysis simultaneously, because the rigid framework of the protein constrains the acidic and basic residues and prevents them from combining with each other, as they would do in solution (Figure 3–49).


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Cell_Biology_Alberts. The fit between an enzyme and its substrate needs to be precise. A small change introduced by genetic engineering in the active site of an enzyme can therefore have a profound effect. Replacing a glutamic acid with an aspartic acid in one enzyme, for example, shifts the position of the catalytic carboxylate ion by only 1 Å (about the radius of a hydrogen atom); yet this is enough to decrease the activity of the enzyme a thousandfold.


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Cell_Biology_Alberts. To demonstrate how enzymes catalyze chemical reactions, we examine an enzyme that acts as a natural antibiotic in egg white, saliva, tears, and other secretions. Lysozyme catalyzes the cutting of polysaccharide chains in the cell walls of bacteria. The bacterial cell is under pressure from osmotic forces, and cutting even a small number of these chains causes the cell wall to rupture and the cell to burst. A relatively small and stable protein that can be easily isolated in large quantities, lysozyme was the first enzyme to have its structure worked out in atomic detail by x-ray crystallography (in the mid-1960s).


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Cell_Biology_Alberts. The reaction that lysozyme catalyzes is a hydrolysis: it adds a molecule of water to a single bond between two adjacent sugar groups in the polysaccharide chain, thereby causing the bond to break (see Figure 2–9). The reaction is energetically favorable because the free energy of the severed polysaccharide chain is lower progress of reaction activation energy for catalyzed reaction Figure 3–48 The rate accelerations caused by five different enzymes.


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Cell_Biology_Alberts. (Adapted from A. Radzicka and R. Wolfenden, Science 267:90–93, 1995.) catalysis. (A) The start of the uncatalyzed reaction that hydrolyzes a peptide bond, with blue shading used to indicate electron distribution in the water and carbonyl bonds. (B) An acid likes to donate a proton (H+) to other atoms. By pairing with the carbonyl oxygen, an acid causes electrons to move away from the carbonyl carbon, making this atom much more attractive to the electronegative oxygen of an attacking water molecule. (C) A base likes to take up H+. By pairing with a hydrogen of the attacking water molecule, a base causes base catalyses electrons to move toward the water oxygen, making it a better attacking group for the carbonyl carbon. (D) By having appropriately than the free energy of the intact chain. However, there is an energy barrier to the positioned atoms on its surface, an enzyme reaction, and a colliding water molecule can break a bond linking two sugars only if the polysaccharide molecule


Cell_Biology_Alberts

Cell_Biology_Alberts. However, there is an energy barrier to the positioned atoms on its surface, an enzyme reaction, and a colliding water molecule can break a bond linking two sugars only if the polysaccharide molecule is distorted into a particular shape—the transition state—in which the atoms around the bond have an altered geometry and electron distribution. Because of this requirement, random collisions must supply a very large activation energy for the reaction to take place. In an aqueous solution at room temperature, the energy of collisions almost never exceeds the activation energy. The pure polysaccharide can therefore remain for years in water without being hydrolyzed to any detectable degree.


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Cell_Biology_Alberts. This situation changes drastically when the polysaccharide binds to lysozyme. The active site of lysozyme, because its substrate is a polymer, is a long groove that holds six linked sugars at the same time. As soon as the polysaccharide binds to form an enzyme–substrate complex, the enzyme cuts the polysaccharide by adding a water molecule across one of its sugar–sugar bonds. The product chains are then quickly released, freeing the enzyme for further cycles of reaction (Figure 3–50).


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Cell_Biology_Alberts. An impressive increase in hydrolysis rate is possible because conditions are created in the microenvironment of the lysozyme active site that greatly reduce the activation energy necessary for the hydrolysis to take place. In particular, lysozyme distorts one of the two sugars connected by the bond to be broken from its normal, most stable conformation. The bond to be broken is also held close to two amino acids with acidic side chains (a glutamic acid and an aspartic acid) that participate directly in the reaction. Figure 3–51 shows the three central steps in this enzymatically catalyzed reaction, which occurs millions of times faster than uncatalyzed hydrolysis.


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Cell_Biology_Alberts. Other enzymes use similar mechanisms to lower activation energies and speed up the reactions they catalyze. In reactions involving two or more reactants, the active site also acts like a template, or mold, that brings the substrates together in the proper orientation for a reaction to occur between them (Figure 3–52A). As we saw for lysozyme, the active site of an enzyme contains precisely positioned can perform both acid catalysis and base catalysis at the same time.


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Cell_Biology_Alberts. Figure 3–50 The reaction catalyzed by lysozyme. (A) The enzyme lysozyme (E) catalyzes the cutting of a polysaccharide chain, which is its substrate (S). The enzyme first binds to the chain to form an enzyme–substrate complex (ES) and then catalyzes the cleavage of a specific covalent bond in the backbone of the polysaccharide, forming an enzyme–product complex (EP) that rapidly dissociates. Release of the severed chain (the products P) leaves the enzyme free to act on another substrate molecule. (B) A space-filling model of the lysozyme molecule bound to a short length of polysaccharide chain before cleavage (Movie 3.8). (B, courtesy of Richard J. Feldmann; PDB code: 3AB6.) This substrate is an oligosaccharide of six sugars, The fnal products are an oligosaccharide of four sugars labeled A through F. Only sugars D and E are shown in detail. (left) and a disaccharide (right), produced by hydrolysis.


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Cell_Biology_Alberts. In the enzyme–substrate complex (ES), the The Asp52 has formed a covalent bond between enzyme forces sugar D into a strained the enzyme and the C1 carbon atom of sugar D. conformation. The Glu35 in the enzyme is The Glu35 then polarizes a water molecule (red ), positioned to serve as an acid that attacks the so that its oxygen can readily attack the C1 adjacent sugar–sugar bond by donating a proton carbon atom and displace Asp52. (H+) to sugar E; Asp52 is poised to attack the C1 carbon atom.


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Cell_Biology_Alberts. atoms that speed up a reaction by using charged groups to alter the distribution of electrons in the substrates (Figure 3–52B). And as we have also seen, when a substrate binds to an enzyme, bonds in the substrate are often distorted, changing the substrate shape. These changes, along with mechanical forces, drive a substrate toward a particular transition state (Figure 3–52C). Finally, like lysozyme, many enzymes participate intimately in the reaction by transiently forming a covalent bond between the substrate and a side chain of the enzyme. Subsequent steps in the reaction restore the side chain to its original state, so that the enzyme remains unchanged after the reaction (see also Figure 2–48). Tightly Bound Small Molecules Add Extra Functions to Proteins