Metabolism Is Composed of Many Coupled Interconnecting Reactions
Hummingbirds are capable of prodigious feats of endurance. For instance, the tiny ruby-throated hummingbird can store enough fuel to fly across the Gulf of Mexico, a distance of some 500 miles, without resting. This achievement is possible because of the ability to convert fuels into the cellular energy currency, ATP, represented by the model at the right. [(Left) K. D. McGraw/Rainbow.] II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Metabolism is essentially a linked series of chemical reactions that begins with a particular molecule and converts it into some other molecule or molecules in a carefully defined fashion (Figure 14.1). There are many such defined pathways in the cell (Figure 14.2), and we will examine a few of them in some detail later. These pathways are interdependent, and their activity is coordinated by exquisitely sensitive means of communication in which allosteric enzymes are predominant (Section 10.1). We will consider the principles of this communication in Chapter 15. We can divide metabolic pathways into two broad classes: (1) those that convert energy into biologically useful forms and (2) those that require inputs of energy to proceed. Although this division is often imprecise, it is nonetheless a useful distinction in an examination of metabolism. Those reactions that transform fuels into cellular energy are called catabolic reactions or, more generally, catabolism. Those reactions that require energy such as the synthesis of glucose, fats, or DNA are called anabolic reactions or anabolism. The useful forms of energy that are produced in catabolism are employed in anabolism to generate complex structures from simple ones, or energy-rich states from energy-poor ones. Some pathways can be either anabolic or catabolic, depending on the energy conditions in the cell. They are referred to as amphibolic pathways. 14.1.1. A Thermodynamically Unfavorable Reaction Can Be Driven by a Favorable Reaction Conceptual Insights, Energetic Coupling, offers a graphical presentation of how enzymatic coupling enables a favorable reaction to drive an unfavorable reaction. How are specific pathways constructed from individual reactions? A pathway must satisfy minimally two criteria: (1) the individual reactions must be specific and (2) the entire set of reactions that constitute the pathway must be thermodynamically favored. A reaction that is specific will yield only one particular product or set of products from its reactants. As discussed in Chapter 8, a function of enzymes is to provide this specificity. The thermodynamics of metabolism is most readily approached in terms of free energy, which was discussed in Sections 1.3.3, 8.2.1, and 8.2.2. A reaction can occur spontaneously only if ∆ G, the change in free energy, is negative. Recall that ∆ G for the formation of products C and D from substrates A and B is given by Thus, the ∆ G of a reaction depends on the nature of the reactant and products (expressed by the ∆ G° term, the standard free-energy change) and on their concentrations (expressed by the second term). An important thermodynamic fact is that the overall free-energy change for a chemically coupled series of reactions is equal to the sum of the freeenergy changes of the individual steps. Consider the following reactions: Under standard conditions, A cannot be spontaneously converted into B and C, because ∆ G is positive. However, the conversion of B into D under standard conditions is thermodynamically feasible. Because free- energy changes are additive, the conversion of A into C and D has a ∆ G° of -3 kcal mol-1 (-13 kJ mol-1), which means that it can occur spontaneously under standard conditions. Thus, a thermodynamically unfavorable reaction can be driven by a thermodynamically favorable reaction to which it is coupled. In this example, the chemical intermediate B, common to both reactions, couples the reactions. Thus, metabolic pathways are formed by the coupling of enzyme-catalyzed reactions such that the overall free energy of the pathway is negative. 14.1.2. ATP Is the Universal Currency of Free Energy in Biological Systems Just as commerce is facilitated by the use of a common currency, the commerce of the cell metabolism is facilitated by the use of a common energy currency, adenosine triphosphate (ATP). Part of the free energy derived from the oxidation of foodstuffs and from light is transformed into this highly accessible molecule, which acts as the free-energy donor in most energy-requiring processes such as motion, active transport, or biosynthesis. ATP is a nucleotide consisting of an adenine, a ribose, and a triphosphate unit (Figure 14.3). The active form of ATP is usually a complex of ATP with Mg2+ or Mn2+ (Section 9.4.2). In considering the role of ATP as an energy carrier, we can focus on its triphosphate moiety. ATP is an energy-rich molecule because its triphosphate unit contains two phosphoanhydride bonds. A large amount of free energy is liberated when ATP is hydrolyzed to adenosine diphosphate (ADP) and orthophosphate (Pi) or when ATP is hydrolyzed to adenosine monophosphate (AMP) and pyrophosphate (PPi). The precise ∆ G° for these reactions depends on the ionic strength of the medium and on the concentrations of Mg2+ and other metal ions. Under typical cellular concentrations, the actual ∆ G for these hydrolyses is approximately -12 kcal mol1 (-50 kJ mol-1). The free energy liberated in the hydrolysis of ATP is harnessed to drive reactions that require an input of free energy, such as muscle contraction. In turn, ATP is formed from ADP and Pi when fuel molecules are oxidized in chemotrophs or when light is trapped by phototrophs. This ATP ADP cycle is the fundamental mode of energy exchange in biological systems. Some biosynthetic reactions are driven by hydrolysis of nucleoside triphosphates that are analogous to ATP namely, guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The diphosphate forms of these nucleotides are denoted by GDP, UDP, and CDP, and the monophosphate forms by GMP, UMP, and CMP. Enzymes can catalyze the transfer of the terminal phosphoryl group from one nucleotide to another. The phosphorylation of nucleoside monophosphates is catalyzed by a family of nucleoside monophosphate kinases, as discussed in Section 9.4.1. The phosphorylation of nucleoside diphosphates is catalyzed by nucleoside diphosphate kinase, an enzyme with broad specificity. It is intriguing to note that, although all of the nucleotide triphosphates are energetically equivalent, ATP is nonetheless the primary cellular energy carrier. In addition, two important electron carriers, NAD+ and FAD, are derivatives of ATP. The role of ATP in energy metabolism is paramount. 14.1.3. ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions How does coupling to ATP hydrolysis make possible an otherwise unfavorable reaction? Consider a chemical reaction that is thermodynamically unfavorable without an input of free energy, a situation common to many biosynthetic reactions. Suppose that the standard free energy of the conversion of compound A into compound B is +4.0 kcal mol-1 (+13 kJ mol-1): The equilibrium constant K eq of this reaction at 25°C is related to ∆ G° (in units of kilocalories per mole) by Thus, net conversion of A into B cannot occur when the molar ratio of B to A is equal to or greater than 1.15 × 10-3. However, A can be converted into B under these conditions if the reaction is coupled to the hydrolysis of ATP. The new overall reaction is Its standard free-energy change of -3.3 kcal mol-1 (-13.8 kJ mol-1) is the sum of the value of ∆ G° for the conversion of A into B [+4.0 kcal mol-1 (+12.6 kJ mol-1)] and the value of ∆ G° for the hydrolysis of ATP [-7.3 kcal mol-1 (-30.5 kJ mol-1)]. At pH 7, the equilibrium constant of this coupled reaction is At equilibrium, the ratio of [B] to [A] is given by The ATP-generating system of cells maintains the [ATP]/[ADP][Pi] ratio at a high level, typically of the order of 500 M1. For this ratio, which means that the hydrolysis of ATP enables A to be converted into B until the [B]/[A] ratio reaches a value of 1.34 × 105. This equilibrium ratio is strikingly different from the value of 1.15 × 10-3 for the reaction A B in the absence of ATP hydrolysis. In other words, coupling the hydrolysis of ATP with the conversion of A into B has changed the equilibrium ratio of B to A by a factor of about 108. We see here the thermodynamic essence of ATP's action as an energy-coupling agent. Cells maintain a high level of ATP by using oxidizable substrates or light as sources of free energy. The hydrolysis of an ATP molecule in a coupled reaction then changes the equilibrium ratio of products to reactants by a very large factor, of the order of 108. More generally, the hydrolysis of n ATP molecules changes the equilibrium ratio of a coupled reaction (or sequence of n reactions) by a factor of 108 . For example, the hydrolysis of three ATP molecules in a coupled reaction changes the equilibrium ratio by a factor of 1024. Thus, a thermodynamically unfavorable reaction sequence can be converted into a favorable one by coupling it to the hydrolysis of a sufficient number of ATP molecules in a new reaction. It should also be emphasized that A and B in the preceding coupled reaction may be interpreted very generally, not only as different chemical species. For example, A and B may represent activated and unactivated conformations of a protein; in this case, phosphorylation with ATP may be a means of conversion into an activated conformation. Such a conformation can store free energy, which can then be used to drive a thermodynamically unfavorable reaction. Through such changes in conformation, molecular motors such as myosin, kinesin, and dynein convert the chemical energy of ATP into mechanical energy (Chapter 34). Indeed, this conversion is the basis of muscle contraction. Alternatively, A and B may refer to the concentrations of an ion or molecule on the outside and inside of a cell, as in the active transport of a nutrient. The active transport of Na+ and K+ across membranes is driven by the phosphorylation of the sodium-potassium pump by ATP and its subsequent dephosphorylation (Section 13.2.1). 14.1.4. Structural Basis of the High Phosphoryl Transfer Potential of ATP As illustrated by molecular motors (Chapter 34) and ion pumps (Section 13.2), phosphoryl transfer is a common means of energy coupling. Furthermore, as we shall see in Chapter 15, phosphoryl transfer is also widely used in the intracellular transmission of information. What makes ATP a particularly efficient phosphoryl-group donor? Let us compare the standard free energy of hydrolysis of ATP with that of a phosphate ester, such as glycerol 3-phosphate: The magnitude of ∆ G° for the hydrolysis of glycerol 3-phosphate is much smaller than that of ATP, which means that ATP has a stronger tendency to transfer its terminal phosphoryl group to water than does glycerol 3-phosphate. In other words, ATP has a higher phosphoryl transfer potential (phosphoryl-group transfer potential) than does glycerol 3phosphate. What is the structural basis of the high phosphoryl transfer potential of ATP? Because ∆ G° depends on the difference in free energies of the products and reactants, the structures of both ATP and its hydrolysis products, ADP and Pi, must be examined to answer this question. Three factors are important: resonance stabilization, electrostatic repulsion, and stabilization due to hydration. ADP and, particularly, Pi, have greater resonance stabilization than does ATP. Orthophosphate has a number of resonance forms of similar energy (Figure 14.4), whereas the γ-phosphoryl group of ATP has a smaller number. Forms like that shown in Figure 14.5 are unfavorable because a positively charged oxygen atom is adjacent to a positively charged phosphorus atom, an electrostatically unfavorable juxtaposition. Furthermore, at pH 7, the triphosphate unit of ATP carries about four negative charges. These charges repel one another because they are in close proximity. The repulsion between them is reduced when ATP is hydrolyzed. Finally, water can bind more effectively to ADP and Pi than it can to the phosphoanhydride part of ATP, stabilizing the ADP and Pi by hydration. ATP is often called a high-energy phosphate compound, and its phosphoanhydride bonds are referred to as high-energy bonds. Indeed, a "squiggle" (~P) is often used to indicate such a bond. Nonetheless, there is nothing special about the bonds themselves. They are high-energy bonds in the sense that much free energy is released when they are hydrolyzed, for the aforegiven reasons. 14.1.5. Phosphoryl Transfer Potential Is an Important Form of Cellular Energy Transformation The standard free energies of hydrolysis provide a convenient means of comparing the phosphoryl transfer potential of phosphorylated compounds. Such comparisons reveal that ATP is not the only compound with a high phosphoryl transfer potential. In fact, some compounds in biological systems have a higher phosphoryl transfer potential than that of ATP. These compounds include phosphoenolpyruvate (PEP), 1,3-bisphosphoglycerate (1,3-BPG), and creatine phosphate (Figure 14.6). Thus, PEP can transfer its phosphoryl group to ADP to form ATP. Indeed, this is one of the ways in which ATP is generated in the breakdown of sugars (Sections 14.2.1, 16.1.6, and 16.1.7). It is significant that ATP has a phosphoryl transfer potential that is intermediate among the biologically important phosphorylated molecules (Table 14.1). This intermediate position enables ATP to function efficiently as a carrier of phosphoryl groups. Creatine phosphate in vertebrate muscle serves as a reservoir of high-potential phosphoryl groups that can be readily transferred to ATP. Indeed, we use creatine phosphate to regenerate ATP from ADP every time we exercise strenuously. This reaction is catalyzed by creatine kinase. At pH 7, the standard free energy of hydrolysis of creatine phosphate is -10.3 kcal mol-1 (-43.1 kJ mol-1), compared with -7.3 kcal mol-1 (-30.5 kJ mol-1) for ATP. Hence, the standard free-energy change in forming ATP from creatine phosphate is -3.0 kcal mol-1 (-12.6 kJ mol-1), which corresponds to an equilibrium constant of 162. In resting muscle, typical concentrations of these metabolites are [ATP] = 4 mM, [ADP] = 0.013 mM, [creatine phosphate] = 25 mM, and [creatine] = 13 mM. The amount of ATP in muscle suffices to sustain contractile activity for less than a second. The abundance of creatine phosphate and its high phosphoryl transfer potential relative to that of ATP make it a highly effective phosphoryl buffer. Indeed, creatine phosphate is the major source of phosphoryl groups for ATP regeneration for a runner during the first 4 seconds of a 100-meter sprint. After that, ATP must be generated through metabolism (Figure 14.7). II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.1. Glucose Metabolism. Glucose is metabolized to pyruvate in 10 linked reactions. Under anaerobic conditions, pyruvate is metabolized to lactate and, under aerobic conditions, to acetyl CoA. The glucose-derived carbons are subsequently oxidized to CO2. II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.2. Metabolic Pathways. [From the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/kegg).] II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.3. Structures of ATP, ADP, and AMP. These adenylates consist of adenine (blue), a ribose (black), and a tri-, di-, or monophosphate unit (red). The innermost phosphorus atom of ATP is designated P , the middle one P , and α the outermost one P . γ β II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.4. Resonance Structures of Orthophosphate. II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.5. Improbable Resonance Structure. The structure contributes little to the terminal part of ATP, because two positive charges are placed adjacent to each other. II. Transducing and Storing Energy 14. Metabolism: Basic Concepts and Design 14.1. Metabolism Is Composed of Many Coupled, Interconnecting Reactions Figure 14.6. High Phosphoryl Transfer Potential Compounds. These compounds have a higher phosphoryl transfer potential than that of ATP and can be used to phosphorylate ADP to form ATP.