Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Introduction to Cell Biology
Macromolecules: Structure, Shape, and Informational Functions
Protein Functions

The chemical properties of protein molecules depend almost entirely on The amino acid residues exposed on their surface, which are capable of forming various weak bonds with other molecules (see Section 3.1.1). For the Interaction of a protein with another molecule (hereinafter referred to as a Ligand) to be effective, many weak bonds must form between them simultaneously. Therefore, only those ligands that fit the protein's surface precisely can bind tightly to it.

The binding site, i.e., the region of a protein that interacts with a ligand, usually has the form of a cavity formed On the surface of the protein molecule by a specific arrangement of Amino Acids. These amino acids often belong to widely separated Regions of the polypeptide chain (Fig. 3-49) and make up only a small fraction of the protein's total amino acids. The remaining Amino acids are necessary to maintain the correct shape of the protein molecule and to create additional binding sites that play a regulatory role. The Role of the protein's interior is usually limited to providing the required surface shape and necessary structural rigidity.

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Fig. 3-49. Hydrogen bonding between the CAP protein and its ligand, cAMP, revealed by X-ray crystallographic Analysis of the complex. The two identical subunits of the dimer are shown to associate to form the binding site (see also Fig. 3-40). (Courtesy of Tom Steitz.)

3.4.1. Protein conformation determines its chemical properties [19, 33]

Neighboring amino acid residues on The surface of a protein molecule often interact in such a way that the reactivity of the side chains of specific amino acids is altered. Such interactions can be divided into several types.

First, neighboring PARTS OF THE polypeptide chain can interact in a way that restricts the access of Water molecules to other regions of the protein surface. Because water molecules tend to form Hydrogen Bonds, they must compete with ligands for the amino acid side chains on the protein surface intended for those ligands. Therefore, the strength of hydrogen bonds (and ionic interactions) between the protein and the ligand is significantly greater if water molecules are excluded. At first glance, it is difficult to imagine a mechanism capable of restricting access of a molecule as small as water to the protein surface without affecting the binding of the ligand itself. However, because of their strong tendency to form hydrogen bonds, water molecules form large molecular networks (Scheme 2-1), and it is often energetically unfavorable for an individual molecule to break away from such a network to penetrate a cavity on the protein surface.

Fig. 3-50. An unusually reactive amino acid in the Active Site of an enzyme. Shown here as an example is the 'charge-Relay system' found in Chymotrypsin, Elastase, and other Serine proteases (see Fig. 3-35). The region of the chain containing aspartic acid induces Histidine to Abstract a proton from serine 195; this activates the serine to form a covalent bond with the enzyme substrate and hydrolyze the peptide bond, as shown in Fig. 3-53.

Second, the clustering of neighboring polar amino acids alters the reactivity of their side chains. For example, a polypeptide chain can fold in such a way that it brings several negatively charged amino acids close together, despite their mutual repulsion. When this occurs, the affinity of each side chain for a positively charged ion increases dramatically. The side chains of Some amino acids can also form Hydrogen bonds and thereby activate normally unreactive side chains (for example, the —CH₂OH group of serine, Fig. 3-50). Activated side chains can participate in reactions that lead to the formation or Cleavage of specific covalent bonds.

Thus, the surface of each protein molecule has unique chemical properties that depend not only on The Nature of the amino acids located on the surface, but also on the precise relative orientation of these amino acids. Therefore, even minor Changes in the conformation of a protein molecule can lead to a dramatic change in its chemical properties.

In cases where the Chemical properties of amino acid side chains cannot solve a specific catalytic problem, Proteins enlist the help of special non-protein molecules. Such ligands often serve as Coenzymes in enzymatic reactions and can be so tightly bound to the protein that they are effectively part of it. Examples include: iron-containing Hemes in Hemoglobin and Cytochromes; thiamine pyrophosphate in Enzymes involved in aldehyde group transfer; biotin in enzymes involved in carboxyl group transfer (see Section 2.4.3). In the course of evolution, Each enzyme has been selected for a specific chemical activity that it exhibits in complex with the protein. Coenzymes are often highly complex organic molecules whose chemical properties in complex with the protein are not always understood in detail. In addition to the reactive center, coenzymes often contain groups that bind them to their respective proteins (Fig. 3-51). Fig. 3-52, A shows space-filling models of two enzymes bound to their coenzymes.

Fig. 3-51. Coenzymes, such as thiamine pyrophosphate (highlighted here in gray), are small molecules that bind to the enzyme's surface, thereby enabling it to catalyze specific reactions. The activity of thiamine pyrophosphate depends on an 'acidic' carbon atom that readily exchanges its bound hydrogen atom for a carbon atom of the substrate molecule. Other parts of the thiamine pyrophosphate molecule apparently serve as 'handles' by which the enzyme holds the coenzyme in the correct position.

3.4.2. Substrate binding is the first step in Enzymatic Catalysis [34]

One of the most important Functions of Proteins is the specific catalysis of Chemical Reactions. In this case, the ligand is a substrate molecule, and its binding by the enzyme is a necessary prerequisite for the chemical reaction (Fig. 3-52, B). Enzymes are capable of enormously accelerating chemical reactions—far more effectively than any artificial catalysts. This high efficiency can be attributed to several factors. First, enzymes increase the local concentration of substrate molecules at the catalytic site and hold the appropriate atoms in the orientation required for the subsequent reaction. Most importantly, however, a portion of the binding energy is directly used for catalysis. This is because substrate molecules, before being converted into reaction products, pass through a series of intermediate forms with altered geometry and electron distribution. The Free energy of all these intermediate forms, and especially of the least stable transition states, is significantly reduced when the molecule is bound to the enzyme surface. Enzymes typically have a much higher affinity for the unstable transition states of substrates than for their stable forms. By utilizing binding energy, enzymes help substrates adopt a specific Transition State, thereby greatly accelerating a single, specific reaction.

Some enzymes interact covalently with one of their substrates. In this process, the substrate becomes bound to an amino acid or a coenzyme molecule. Such enzymatic reactions often occur in several steps, where one substrate is captured by the binding site and covalently bound, and then reacts on the enzyme surface with a second substrate (Fig. 3-53). By the end of each reaction cycle, the free enzyme is regenerated.

Fig. 3-52. Computer models. A. Cytochrome c with its prosthetic group, heme. B. Egg white Lysozyme with bound oligosaccharide. In both cases, the bound ligand is shown in color. (Courtesy of Richard J. Feldmann.)

Fig. 3-53. Some enzymes form a transient covalent bond with their substrates. In the example shown here, the carboxyl group of a cleaved polypeptide chain forms a covalent bond with an activated serine residue of a protease. After dissociation of the unbound part of the polypeptide, a second reaction step (not shown here) occurs: a water molecule hydrolyzes the newly formed covalent bond and releases the remaining part of the polypeptide chain, allowing serine 195 to participate in the next reaction cycle (see also Fig. 3-50).

The METABOLISM/10.html">Mechanism of enzyme Action imposes a limit on the number of substrate molecules that can be 'processed' by a single enzyme molecule per unit of time. As the Substrate Concentration increases, The rate of product formation initially also increases up to a maximum value (Fig. 3-54). At this point, the enzyme molecules are saturated with substrate, and the reaction rate (denoted as V_max) now depends only on how rapidly the enzyme can process a single substrate molecule. The ratio of this rate to the Enzyme Concentration is called the turnover number, which for many enzymes is about 1000 substrate molecules per second, but in exceptional cases can reach values of 10⁶ or more.

Another kinetic parameter frequently used to characterize enzymes is their Michaelis constant, K_m, defined as the substrate concentration at which the reaction rate is half-maximal (Fig. 3-54). A low K_m value indicates that the enzyme reaches its maximum rate of catalysis at a low substrate concentration and usually corresponds to very tight binding of the substrate by the enzyme.

3.4.3. Enzymes accelerate reactions but do not shift chemical equilibrium

No matter how sophisticated an enzyme may be, it cannot make the reaction it catalyzes more energetically favorable. It cannot alter the free-energy difference between the starting substrate and the final product of the reaction. Like the simple binding discussed earlier, every chemical reaction has an equilibrium point at which the rates of the forward and reverse reactions are equal and, consequently, no further change in concentrations occurs (see Fig. 3-7). If an enzyme accelerates the forward reaction A + B → AB by 10⁸-fold, it must also accelerate the reverse reaction AB → A + B by 10⁸-fold. The ratio of the forward and reverse reaction rates depends only on the concentrations of A, B, and AB. The equilibrium position remains exactly the same regardless of whether the enzyme catalyzes the reaction or not.

Fig. 3-54. As the substrate concentration increases, the rate of the enzymatic reaction V increases until it reaches its maximum value V_max. This occurs at a substrate concentration at which no unoccupied enzyme molecules remain, and the reaction rate is limited by the rate of the catalytic process on the enzyme surface. For most enzymes, the substrate concentration at which the reaction rate is half-maximal, K_m, reflects the strength of substrate binding to the enzyme. High K_m values correspond to weak binding, and vice versa.

3.4.4. Many enzymes drive reactions preferentially in one direction by coupling them to ATP Hydrolysis [35].

A living cell is a chemical system far from equilibrium: the product of each enzyme is usually rapidly consumed because it serves as a substrate for another enzyme in the same metabolic pathway. Even more importantly, many of the enzymatic reactions already described in Chapter 2 are coupled to the cleavage of ATP into ADP and inorganic phosphate (see Section 2.4.2). For this to be possible, the ATP pool must in turn be maintained at a level far from equilibrium, so that the ratio of ATP concentration to the concentration of its hydrolysis products remains high. Thus, the ATP pool serves as a "battery" that maintains a constant transfer of energy and atoms within The Cell along metabolic pathways determined by the available enzymes. The approach of a living system to chemical equilibrium is equivalent to its decay and death.

3.4.5. Multienzyme complexes increase the rate of cellular metabolism [36]

The ability of enzymes to accelerate chemical reactions is crucial for sustaining life. Indeed, a cell must resist the inevitable process of decay, which keeps it in a state far from chemical equilibrium. If the rates of key forward reactions were not higher than those of their reverse reactions, the cell would quickly die. An idea of the metabolic rate can be gained from the fact that the ATP pool of a typical mammalian cell is completely turned over every 1–2 minutes (i.e., all molecules are broken down and replaced by newly synthesized ones). This means that in a single second, each cell consumes 10⁷ molecules of ATP, and the entire human body thus processes about a gram of ATP per minute.

Such high rates of cellular reactions are made possible by the efficiency of enzyme catalysts. Indeed, the efficiency of many Key Enzymes is so high that any further increase would be pointless, because the reactions they catalyze are limited by the rate of collision between the enzyme and its substrates; in other words, the reaction rates are diffusion-limited.

If a reaction is diffusion-limited, its rate will depend on the concentrations of both the enzyme and the substrate. Therefore, to achieve a very high rate for a series of sequential reactions, each intermediate and all the enzymes must be present in high concentrations. However, the vast number of different reactions occurring simultaneously in a cell limits the achievable concentrations of reactants. In reality, most metabolites are present in micromolar concentrations (10-⁶ M), and the cellular concentration of most enzymes is much lower. How, then, is it possible to maintain such extremely high metabolic rates?

The answer lies in the Spatial Organization of cellular components. Reaction rates can be increased without increasing substrate concentrations by organizing the various enzymes involved in sequential reactions into a large multienzyme complex. In this type of organization, the product of enzyme A is passed directly to enzyme B, and so on, until the final product is formed, eliminating the rate-limiting diffusion step even at very low intracellular concentrations of intermediates. Such enzyme complexes are very common. The Structure of one of them—Pyruvate dehydrogenase—was shown in Fig. 2-40. These complexes are involved in almost all aspects of metabolism, including the central genetic processes of DNA, RNA, and Protein Synthesis. While it is possible that a small number of eukaryotic enzymes diffuse freely in solution, most appear to have evolved binding sites that concentrate them with Other Enzymes of related function in specific regions of the cell, thereby increasing the rate and efficiency of the reactions they catalyze.

Fig. 3-55. A large increase in the concentration of interacting molecules can be achieved by confining them to a membrane-bounded compartment within a Introduction/5.html">Eukaryotic Cell.

Cells also have another way of increasing the rate of metabolic reactions, which involves intracellular membranes.

3.4.6. Intracellular membranes accelerate diffusion-limited reactions [37]

The extensive network of intracellular membranes in Eukaryotic cells accelerates reactions that would otherwise be diffusion-limited in at least two ways. First, membranes can confine a set of substrates and the enzymes that act on them within a single compartment, such as The Endoplasmic reticulum or The Nucleus. Assuming that each such compartment occupies about 10% of the cell volume, the concentration of reactants within the compartment can be 10 times higher than in an equivalent cell without compartmentalization (Fig. 3-55).

The second way membranes can increase reaction rates is by restricting the diffusion of reactants to the two dimensions of the membrane surface itself. Enzymes and their substrates confined to two dimensions will collide with each other much more frequently than they would during three-dimensional diffusion, even though the rate of molecular diffusion in a membrane is about 100 times slower than in an aqueous solution (Fig. 3-56). This process is likely used by enzymes and substrates involved in the synthesis of lipid molecules, where the substrates are dissolved directly in The Lipid Bilayer. It may also be used to accelerate many other reactions involving membrane-bound enzymes.

A similar mechanism of "Facilitated Diffusion" has been found to increase the rate at which certain regulatory proteins locate the Gene-specific DNA sequences they bind to directly on the chromosome. These proteins have a weak affinity for all DNA sites. They constantly collide with the chromosome, "slide" along it, and in this way scan the entire length of the DNA until they find their specific binding sites.

Fig. 3-56. Reaction rates increase when three-dimensional diffusion is replaced by two-dimensional diffusion due to the presence of membranes. Shown here is the result of a series of theoretical calculations. A. In the absence of membranes, an average molecule would take about 30 min to find any single "target" inside a spherical particle 10 µm in diameter. B. If the target is fixed on a membrane, the diffusion time is significantly reduced. An average molecule takes about 1 s to reach a large internal membrane and about 2 min to find the target on the membrane. C. If the area of the internal membrane is reduced tenfold, the molecule will take 10 s to reach the membrane, but searching for the target will now take approximately 10 times less time than in case B. Thus, the collision efficiency in case C is nearly 100 times higher than in A.

3.4.7. Protein molecules can reversibly change their shape [38]

In general, natural Selection has favored the evolution of Polypeptides that adopt specific, stable Conformations. However, some protein molecules—perhaps even most of them—have two or more slightly different conformations and can alter their function by switching reversibly between them. In such allosteric protein, for example, several different sets of hydrogen bonds of roughly equal energy can form, with each set requiring different spatial relationships between two regions of the polypeptide chain. The alternative stable conformations are typically separated by unstable intermediate states, so that the molecule "flickers" between stable conformations.

Each discrete conformation of an allosteric protein has a slightly different surface and, consequently, a different capacity to interact with other molecules. Often, only one of the two conformations has a high affinity for a particular ligand; in this case, the presence or absence of the ligand determines the conformation adopted by the protein (Fig. 3-57). When two different ligands can bind to different sites on the surface of the same protein, A change in the concentration of one ligand alters the protein's affinity for the other. Such allosteric changes play a key role in The regulation of many biological processes.

3.4.8. Allosteric proteins are involved in the REGULATION OF METABOLISM [39]

Allosteric proteins participate in feedback regulation, which controls the flux of metabolites through metabolic pathways (see Section 2.5). For example, enzymes acting at the Cytology/cytology/16.html">Early stages of a metabolic pathway are almost always allosteric proteins capable of existing in two alternative conformations. One of these is the active conformation. In this active state, the protein binds the substrate at its active site and converts it into the next metabolite in the pathway. The other conformation is inactive. In this state, the protein tightly binds the end product of the same pathway at a specific surface site (the regulatory site). As the end product accumulates, it binds to the enzyme and shifts it into the inactive conformation (negative feedback), which becomes stable because the product can only bind the enzyme in this form. In other cases, an enzyme in a metabolic pathway is activated by an allosteric transition that occurs when the cell is deficient in the pathway's product, prompting the enzyme to bind an accumulating ligand. In this scenario, the ligand binds to the active form of the enzyme (positive feedback), and this binding requires a transition from the inactive to the active conformation (see Fig. 3-57). The result of positive and negative feedback regulation is that a given product is synthesized in the cell only when needed, thereby maintaining relatively constant concentrations of all metabolites.

Fig. 3-57. Each conformation of an allosteric protein can be stabilized by the preferential binding of a ligand. Tight binding of a ligand to only one of the possible conformations of an allosteric protein drives the protein into that conformation. Thus, a high concentration of ligand X will activate the protein shown, whereas a high concentration of ligand Y will inactivate it.

Figure 3-58. Diagram showing how the conformation of one subunit affects the conformation of neighboring subunits in a symmetric protein composed of identical allosteric subunits. The binding of a single regulatory ligand molecule to one subunit alters the conformation of that subunit, as shown in Figure 3-57. Because this change promotes a tightly bound conformation, the binding of the first ligand molecule increases the affinity of the other subunits for binding the same ligand. Thus, the enzyme can be activated by a relatively small increase in the concentration of the regulatory ligand (see Figure 3-59).

3.4.9. Allosteric Proteins are essential for cell signaling [40]

We have already noted that allosteric proteins (such as those involved in feedback regulation) have at least two binding sites—one for the substrate and one or more for regulatory ligands. These sites occupy different regions of the protein surface, and the recognized ligands can be completely different. Because the binding of a ligand to its respective site can affect another site by changing the protein's conformation, any metabolic process in the cell can be regulated by the product of any other reaction, regardless of its chemical nature. For example, Glycogen Synthesis and Breakdown in Muscle cells are regulated by the concentration of bound Ca²⁺ via allosteric enzymes whose activity changes with variations in cytosolic Ca²⁺ concentration (see Section 12.4.4).

Allosteric proteins are particularly sensitive to signals if, as is often the case, they function cooperatively as identical subunits in a symmetric assembly. In such proteins, a conformational change in one subunit induced by ligand binding can help neighboring subunits bind the same ligand (Figure 3-58). As a result, a relatively small change in the environmental ligand concentration triggers the transition of the entire assembly from an inactive to an active conformation, or vice versa. If the ligand binds preferentially to the active conformation of each enzyme subunit, this will lead to a sharp increase in enzymatic activity as the ligand concentration rises (Figure 3-59). The structure of one well-studied allosteric enzyme, aspartate transcarbamoylase, is shown in Figure 3-60.

3.4.10. Proteins can be made to change their conformation [40, 41]

Proteins drive the directional flow of all cellular processes. How, then, can protein molecules themselves be made to move in an orderly fashion? Before answering this question, we must consider how the cell controls Conformational Changes in allosteric proteins. Let us consider an allosteric protein capable of adopting two alternative conformations: an inactive, low-energy state K and an active, high-energy state K*, whose energies differ by 4.3 kcal/mol (which roughly corresponds to the energy of forming four hydrogen bonds on the protein surface). With such an energy difference, the probability of conformation K will be 1000 times greater than that of conformation K* (Table 3-3), and the protein will almost always reside in the inactive conformation. There are, however, two ways to force the protein into the active conformation.

Figure 3-59. With increasing ligand concentration, the activity of the multi-subunit allosteric enzyme shown in Figure 3-58 will produce a "sigmoidal" curve (colored curve) due to the Cooperative binding of ligand molecules. In contrast, the Activation of a single-subunit allosteric enzyme is described by a simple saturation curve (black curve). The dashed line shows the maximum level of activity reached at very high ligand concentrations, which is the same in both cases.

The binding of a small-molecule ligand, metaphorically speaking, "pulls" the molecule into the active K* conformation. If the ligand binds only to K*, the energy of this conformation is selectively decreased, while the energy of K remains unchanged. Because the ligand binds to the protein relatively weakly (most of the binding energy is spent on maintaining the protein shape suitable for the ligand), it dissociates easily, making this conformational change completely reversible.

The other way is to use additional chemical energy to "push" the protein from the K conformation to the active K* conformation. In this case, the conformational change is almost irreversible. Typically, this involves the covalent transfer of a phosphate group from an ATP molecule to serine, Threonine, or Tyrosine residues of the protein, forming a covalent bond. Suppose this phosphorylation reaction, driven by the favorable hydrolysis of ATP to ADP, creates a charge repulsion that is unfavorable for the K conformation. If this repulsion is reduced in the active K* form, the transition from K to K* will be greatly facilitated by phosphorylation (Figure 3-61). Regulated phosphorylation, which activates or inhibits the function of specific proteins, is a common phenomenon in eukaryotic cells (see Section 3.2.3); indeed, approximately one-tenth of the various proteins in mammalian cells contain covalently bound phosphate.

Figure 3-60. The enzyme aspartate transcarbamoylase is turned off in response to the binding of cytosine triphosphate (CTP). The enzyme complex consists of six catalytic subunits and six Regulatory Subunits. The structures of its inactive and active forms have been determined by X-ray crystallography. Each regulatory subunit can bind one molecule of CTP, which is one of the final products of the pathway. This pathway begins when the enzyme catalyzes The formation of carbamoyl aspartate from carbamoyl phosphate and aspartic acid. Through this negative feedback regulation, the enzyme is prevented from producing more CTP than the cell requires. (Adapted from K. L. Krause, K. W. Volz, and W. N. Lipscomb, Proc. Natl. Acad. Sci. USA 82: 1643–1647, 1985.)

Figure 3-61. Phosphorylation by ATP can activate an allosteric protein. In this example, the inactive conformation of the unphosphorylated protein A is 1000 times more energetically favorable due to a free-energy difference of 4.3 kcal/mol (see Table 3-3). When it is phosphorylated, the active conformation of protein B is 100 times more favorable (2.8 kcal/mol) because phosphorylation creates an energetically unfavorable charge repulsion; this effect is partially relieved by the transition to the active conformation K*. Consequently, phosphorylation "pushes" the enzyme into the active conformation. Alternatively, phosphorylation can lead to charge attraction that brings two distant parts of an allosteric protein closer together.

Sometimes, when ADP is added to such phosphorylated proteins in vitro, ATP synthesis is observed. These data directly demonstrate that a significant portion of the energy of ATP hydrolysis was stored in the strained conformation adopted by the protein upon its phosphorylation. How, then, do energy-consuming conformational changes in a protein drive movement and perform useful work in the cell?

3.4.11. Energy-driven conformational changes in proteins can be used to perform useful work [42]

Suppose a protein needs to "walk" along a thin filament, such as an Actin filament or a DNA molecule. Figure 3-62 shows how an allosteric protein can accomplish this task by adopting different conformations. If nothing directs or orders these conformational changes, the changes in the protein's shape will be completely reversible; that is, the protein will wander randomly and aimlessly back and forth along the filament or fiber.

Because work is performed during directional protein movement, the Laws of Thermodynamics dictate that some energy must be expended to drive this motion (otherwise, this movement could be used to create a perpetual motion machine). Therefore, no matter how we modify the model shown in Figure 3-62—for example, by introducing ligands that stabilize one conformation or another—the protein molecule will not be capable of directional movement unless it is provided with an energy source.

The sequence of conformational changes in the protein must somehow be made directional. For example, the entire cycle can become directional if one of the steps is made irreversible. One way to achieve irreversibility is to use the previously described phosphorylation-dephosphorylation cycle. However, allosteric changes in Proteins can also be driven without this, by utilizing the energy of ATP hydrolysis. For instance, in the modified cyclic movement scheme shown in Figure 3-63, the binding of ATP forces the protein to change from conformation 1 to conformation 2. ATP hydrolysis then occurs, yielding bound ADP and inorganic phosphate (P_i). This hydrolysis is accompanied by the transition from conformation 2 to conformation 3. Finally, the release of ADP and P_i allows the protein to return to conformation 1.

Figure 3-62. Schematic representation of a "walking" allosteric protein. Although three different conformations of the protein allow it to move both backward and forward along the fiber to which it is bound, continuous movement in a single direction is impossible.

Because the sequence of conformational transitions 1 → 2 → 3 → 1 expends the energy of ATP hydrolysis, the entire cycle becomes virtually irreversible under physiological conditions (i.e., the probability of ATP synthesis from ADP and P_i via the pathway 1 → 3 → 2 → 1 is extremely low). Since irreversibility ensures the directionality of the cycle, the protein molecule in our schematic example will continuously move to the right. Examples of proteins that undergo directional movement via this mechanism include the muscle protein Myosin and DNA helicase, which plays a key role in DNA Replication.

Many protein machines use similar mechanisms to perform orderly movements. All of these proteins are capable of undergoing cyclic changes in shape driven by ATP hydrolysis. Some of them are transiently phosphorylated during the cycle, while others are not.

3.4.12. Membrane allosteric proteins, utilizing the energy of ATP, can serve as molecular pumps [43]

Allosteric proteins can use the energy of ATP hydrolysis not only to generate mechanical force, but also to perform Other forms of work, such as pumping specific ions into or out of the cell. For example, an allosteric protein present in The Plasma Membrane of all animal cells, called the (Na⁺, K⁺)-dependent ATPase, pumps 3 Na⁺ ions out of the cell and 2 K⁺ ions into the cell during each cycle of conformational changes accompanied by ATP-dependent protein phosphorylation (see Section 6.4.5). This pump, driven by ATP energy, consumes more than 30% of the Energy Requirements of most cells. The continuous pumping of Na⁺ out and K⁺ in results in an intracellular concentration of Na⁺ that is lower, and a K⁺ concentration that is higher, than in the extracellular medium. In this way, oppositely directed transmembrane concentration gradients of K⁺ and Na⁺ ions are established. The energy stored in these and other ion gradients, in turn, drives conformational changes in many other membrane allosteric proteins, enabling them to perform useful work for the cell.

3.4.13. Proteins can harness the energy of ion gradients to perform useful work [43, 44]

ATP and other nucleoside triphosphates are extremely important, but not the only, Energy Sources for proteins to perform useful work. An ion gradient across various cellular membranes can store and expend energy much like a HEAD of water on opposite sides of a dam. For example, the large Na⁺ concentration gradient across the plasma membrane generated by the (Na⁺, K⁺)-dependent ATPase drives other protein pumps that transport glucose or specific amino acids into the cell.

Fig. 3-63. A "walking" allosteric protein in which the transition between three conformations is driven by the hydrolysis of a bound ATP molecule. The cycle becomes virtually irreversible because one of these transitions is coupled to ATP hydrolysis. Through repeated cycles, the protein moves continuously to the right along the filament.

Membrane allosteric pumps driven by the energy of ATP hydrolysis can operate in reverse and use the energy of an ion gradient to synthesize ATP. Indeed, as we will see in Chapter 7, it is this mechanism that harnesses the energy of the proton gradient [H+] (directed across The inner mitochondrial membrane) in animals to synthesize most ATP molecules.

3.4.14. Protein machines play a major role in many biological processes [45]

Complex cellular processes, such as DNA replication or protein synthesis, are carried out by multienzyme complexes that function as sophisticated "protein machines." For example, the numerous Proteins of the DNA replication machinery move coordinately relative to one another, enabling the entire complex to move rapidly along the DNA like a zipper (see Section 5.3.7).

In such protein machines, the hydrolysis of bound nucleoside triphosphate molecules drives directional conformational changes in individual proteins, causing a group of these proteins to move coordinately. In this way, the appropriate enzymes move directly to the site where they are needed to perform a specific reaction, rather than waiting for random collisions between individual reaction components. A simple mechanical analogy that captures The Essence of these "high-tech" solutions to cellular needs is illustrated in Fig. 3-64. Apparently, most of the major processes occurring in cells are carried out by such highly complex, multicomponent protein machines.

Summary

The BIOLOGICAL FUNCTIONS OF a protein are determined by the details of the chemical properties of its surface. Cavities on the protein surface, formed by precisely positioned amino acid residues, constitute specific binding sites. Enzymes catalyze chemical changes in their bound substrate molecules; to expand their capabilities, they often employ small, tightly bound coenzyme molecules. The Rate of Enzymatic reactions is frequently diffusion-limited, but it can be higher if the enzyme and substrate are brought together within the same small cellular compartment.

The binding of ligands to the surface of allosteric proteins reversibly alters their shape. Changes induced by the binding of one ligand can affect the binding of a second ligand, providing a mechanism for regulating various cellular processes. The input of additional chemical energy can drive directional changes in protein shape. For example, by coupling allosteric changes to ATP hydrolysis, proteins can perform useful work, such as generating mechanical force or pumping ions across a membrane. Highly efficient "protein machines" can also be formed by linking coordinately functioning proteins into multienzyme complexes. It is likely that such Protein Assemblies carry out many of the major biological reactions.

Fig. 3-64. A "protein machine." Protein Complexes often consist of one or more subunits capable of movement driven by an energetically favorable change upon binding a substrate molecule (see Fig. 3-63). Such protein movements are particularly useful to the cell if they occur within a large protein complex in which, as illustrated, the activities of the different subunits are coordinated.

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