Energy, Enzymes, and Metabolism

Chemical equilibrium and free energy

Every chemical reaction proceeds to a certain extent, but not necessarily to completion. In other words, all the reactants present are not necessarily converted to products. Each reaction has a specific equilibrium point, and that equilibrium point is related to the free energy released by the reaction under specified conditions. To understand the principle of equilibrium, consider the following example. Most cells contain glucose 1-phosphate, which is converted in the cell to glucose 6-phosphate. Imagine that westart out with an aqueous solution of glucose 1-phosphate that has a concentration of 0.02 M. (M stands for molar concentration. The solution is maintained under constant environmental conditions (25°C and pH 7). As the reaction proceeds slowly to equilibrium, the concentration of the product, glucose 6-phosphate, rises from 0 to 0.019 M, while the concentration of the reactant, glucose 1-phosphate, falls to 0.001 M. At this point, equilibrium is reached. From then on, the reverse reaction, from glucose 6-phosphate to glucose 1-phosphate, progresses at the same rate as the forward reaction.

At equilibrium, then, this reaction has a product-to-reactant ratio of 19:1 (0.019/0.001), so the forward reaction has gone 95 percent of the way to completion (“to the right,” as written). Therefore, the forward reaction is an exergonic reaction. This result is obtained every time the experiment is run under the same conditions. The reaction is described by the equation glucose 1-phosphate ~ glucose 6-phosphate The change in free energy (DG) for any reaction is related directly to its point of equilibrium. The further toward completion the point of equilibrium lies, the more free energy is given off. In an exergonic reaction, such as the conversion of glucose 1-phosphate to glucose 6-phosphate, DG is a negative number (in this example, DG = –1.7 kcal/mol, or –7.1 kJ/mol). Alarge, positive DG for a reaction means that it proceeds hardly at all to the right (A→B). But if the product is present, such a reaction runs backward, or “to the left” (A←B), (nearly all B is converted to A). A DG value near zero is characteristic of a readily reversible reaction: reactants and products have almost the same free energies. The principles of thermodynamics we have been discussing apply to all energy exchanges in the universe, so they are very powerful and useful. Next, we’ll apply them to reactions in cells that involve the biological energy currency, ATP.

ATP: Transferring Energy in Cells

All living cells rely on adenosine triphosphate, or ATP, for the capture and transfer of the free energy needed to do chemical work and maintain the cells. ATP operates as a kind of energy currency. That is, just as you may earn money from a job and then spend it on a meal, some of the free energy released by certain exergonic reactions is captured in ATP which can then release free energy to drive endergonic reactions. ATP is produced by cells in a number of ways (which we will describe in the next two chapters) and it is used in many ways. ATP is not an unusual molecule. In fact, it has another important use in the cell: it can be converted into a building block for DNA and RNA. But two things about ATP make it especially useful to cells: it releases a relatively large amount of energy when hydrolyzed and it can phosphorylate (donate a phosphate group to) many different molecules. We will examine these two properties in the discussion that follows.

ATP hydrolysis releases energy

An ATP molecule consists of the nitrogenous base adenine bonded to ribose (a sugar), which is attached to a sequence of three phosphate groups. The hydrolysis of ATP yields ADP (adenosine diphosphate) and an inorganic phosphate ion (abbreviated Pi, short for HPO₄^2–), as well as free energy:

ATP + H₂O →ADP + Pi + free energy

The important property of this reaction is that it is exergonic, releasing free energy. The change in free energy (DG) is about –12 kcal/mol (–50 kJ/mol) at the temperature, pH and substrate concentrations typical of living cells.

The free energy of the P—O bond between phosphate groups is much higher than the energy of the H—O bond that forms after hydrolysis. So some usable energy is released upon hydrolysis. Because phosphates are negatively charged and so repel each other, it takes energy to get phosphates near enough to each other to make the covalent bond that links them together (e.g., to add a phosphateto ADP to make ATP).

An active cell requires millions of molecules of ATP per second to drive its biochemical machinery. An ATP moleculeis consumed within a second following its formation, on average. At rest, an average person hydrolyzes and produces about 40 kg of ATP per day—as much as some people weigh. This means that each ATP molecule undergoes about 10,000 cycles of synthesis and hydrolysis every day.

Enzymes: Biological Catalysts

The reactions that occur in cells are so slow that they could not contribute to life unless the cells did something to speed them up. That is the role of catalysts: substances that speed up a reaction without being permanently altered by that reaction. Acatalyst does not cause a reaction that would not take place eventually without it, but merely speeds up the rates of both forward and backward reactions, allowing equilibrium to be approached faster. Most biological catalysts are proteins called enzymes. Although we will focus here on proteins, some catalysts — perhaps the earliest ones in the origin of life — are RNA molecules called ribozymes. A biological catalyst, whether protein or RNA, is a framework or scaffold in which chemical catalysis takes place. It does not matter whether the framework is RNA or protein — indeed, artificial catalysts can be made from DNA. Evolution has selected proteins as catalysts, probably because of their great diversity in three-dimensional structure and variety of chemical functions.

For a reaction to proceed, an energy barrier must be overcome

An exergonic reaction may release a great deal of free energy, but the reaction may take place very slowly. Some reactions are slow because there is an energy barrier between reactants and products. Think about a gas stove. The burning of the natural gas (methane + O₂→CO₂ + H₂O) is obviously an exergonic reaction—heat and light are released. Once started, the reaction goes to completion: all of the methane reacts with oxygen to form carbon dioxide and water vapor. Because burning methane liberates so much energy, it might expects this reaction to proceed rapidly whenever methane is exposed to oxygen. But this does not happen. Simply mixing methane with air produces no reaction. Methane will start burning only if a spark — an input of energy — is provided. (On the stove, this energy is supplied by electricity.) The need for this spark to start the reaction shows that there is an energy barrier between the reactants and the products. In general, exergonic reactions proceed only after the reactants are pushed over the energy barrier by a small amount of added energy. The energy barrier thus represents the amount of energy needed to start the reaction, known as the activation energy (Ea). In a chemical reaction, the activation energy is the energy needed to change the reactants into unstable molecular forms called transition-state species. Transition-state species have higher free energies than either the reactants or the products. Their bonds may be stretched and hence unstable. Although the amount of activation energy needed for different reactions varies, it is often small compared with the change in free energy of the reaction. The activation energy that startsa reaction is recovered during the ensuing “downhill” phase of the reaction, so it is not a part of the net free energy change, DG. Where does the activation energy come from? In any collection of reactants at room or body temperature, molecules are moving around and could use their kinetic energy of motion to overcome the energy barrier, enter the transition state and react. However, at normal temperatures, only a few molecules have enough energy to do this; most have insufficient kinetic energy for activation, so the reaction takes place slowly. If the system were heated, all the reactant molecules would move faster and have more kinetic energy. Since more of them would have energy exceeding the required activation energy, the reaction would speed up. However, adding enough heat to increase the average kinetic energy of the molecules won’t work in living systems. Such a nonspecific approach would accelerate all reactions, including destructive ones, such as the denaturation of proteins. A more effective way to speed up a reaction in a living system is to lower the energy barrier. In living cells, enzymes accomplish this task.

Enzymes bind specific reactant molecules

Catalysts increase the rate of chemical reactions. Most nonbiological catalysts are nonspecific. For example, powdered platinum catalyzes virtually any reaction in which molecular hydrogen (H2) is a reactant. In contrast, most biological catalysts are highly specific. These complex molecules of protein (enzymes) or RNA (ribozymes) catalyze relatively simple chemical reactions. An enzyme or ribozyme usually recognizes and binds to only one or a few closely related reactants and it catalyzes only a single chemical reaction. In the discussion that follows, we focus on enzymes but you should note that similar rules of chemical behavior apply to ribozymes as well.

In an enzyme-catalyzed reaction, the reactants are called substrates. Substrate molecules bind to a particular site on the enzyme, called the active site, where catalysis takes place. The specificity of an enzyme results from the exact three-dimensional shape and structure of its active site, into which only a narrow range of substrates can fit. Other molecules — with different shapes, different functional groups, and different properties — cannot properly fit and bind to the active site. The names of enzymes reflect the specificity of their functions and often end with the suffix “-ase.” For example, the enzyme RNA polymerase catalyzes the formation of RNA, but not DNA and the enzyme hexokinase accelerates the phosphorylation of hexose sugars, but not pentose sugars. The binding of a substrate to the active site of an enzyme produces an enzyme–substrate complex (ES) held together by one or more means, such as hydrogen bonding, ionic attraction, or covalent bonding. The enzyme–substrate complex gives rise to product and free enzyme:

E + S →ES →E + P,

where E is the enzyme, S is the substrate, P is the product, and ES is the enzyme–substrate complex. The free enzyme (E) is in the same chemical form at the end of the reaction as at the beginning. While bound to the substrate, it may change chemically but by the end of the reaction it has been restored to its initial form.

Enzymes lower the energy barrier but do not affect equilibrium

When reactants are part of an enzyme–substrate complex, they require less activation energy than the transition-state species of the corresponding uncatalyzed reaction. Thus the enzyme lowers the energy barrier for the reaction— it offers the reaction an easier path. When an enzyme lowers the energy barrier, both the forward and the reverse reactions speed up, so the enzyme-catalyzed overall reaction proceeds toward equilibrium more rapidly than the uncatalyzed reaction. The final equilibrium (and DG) is the same with or without the enzyme. Adding an enzyme to a reaction does not change the difference in free energy (DG) between the reactants and the products. It does change the activation energy and, consequently, the rate of reaction. For example, if 600 molecules of a protein with arginine as its terminal amino acid just sit in solution, the proteins tend toward disorder, and the terminal peptide bonds break, releasing the arginines (DS increases). After 7 years, about half (300) of the proteins will have undergone this reaction. With the enzyme carboxypeptidase A catalyzing the reaction, however, the 300 arginines are released in half a second! After formation of the enzyme–substrate complex, chemical interactions occur. These interactions contribute directly to the breaking of old bonds and the formation of new ones.

Enzymes induce strain in the substrate

Once a substrate has bound to the active site, the enzyme can cause bonds in the substrate to stretch, putting it in an unstable transition state. For example, the polysaccharide substrate for the enzyme lysozyme enters the active site in a flat-ringed “chair” shape, but the active site quickly causes it to flatten out into a “sofa”. The resulting stretching of its bonds causes them to be less stable and more reactive to the enzyme’s other substrate, water.

Enzymes temporarily addchemical groupes to substrate

The side chains (R groups) of an enzyme’s amino acids may be direct participants in making its substrates more chemically reactive. For example, in acid-base catalysis, the acidic or basic side chains of the amino acids forming the active site may transfer H+ to or from the substrate, destabilizing a covalent bond in the substrate and permitting it to break. In covalent catalysis, a functional group in a side chain forms a temporary covalent bond with a portion of the substrate. In metal ion catalysis, metal ions such as copper, zinc, iron and manganese, which are firmly bound to side chains of the protein, can lose or gain electrons without detaching from the protein.

Molecular structure determines enzyme function

Most enzymes (and ribozymes) are much larger than their substrates. An enzyme is typically a protein containing hundreds of amino acids, and may consist of a single folded polypeptide chain or several subunits. Its substrate is generally a small molecule. The active site of the enzyme is usually quite small, not more than 6–12 amino acids.

The active site is specific to the substrate

The remarkable ability of an enzyme to select exactly the right substrate depends on a precise interlocking of molecular shapes and interactions of chemical groups at the binding site. The binding of the substrate to the active site depends on the same kinds of forces that maintain the tertiary structure of the enzyme: hydrogen bonds, the attraction and repulsion of electrically charged groups, and hydrophobic interactions. In 1894, the German chemist Emil Fischer compared the fit between an enzyme and its substrate to that of a lock and key. Fischer’s model persisted for more than half a century with only indirect evidence to support it. The first direct evidence came in 1965, when David Phillips and his colleagues at the Royal Institution in London succeeded in crystallizing the enzyme lysozyme and determined its tertiary structure using the techniques of X-ray crystallography. They observed a pocket in lysozyme that neatly fits its substrate.

An enzyme changes shape when it binds a substrate

As proteins, enzymes are not immutable structures. Just as the structure of egg white protein changes when the egg is heated, many enzymes change their structure (albeit less dramatically) when they bind to their substrates. These shape changes expose those regions of the enzyme — the active sites — that actually react with the substrate. Such a change in enzyme shape caused by substrate binding is called induced fit. Induced fit can be observed in the enzyme hexokinase, when it is studied with and without one of its substrates, glucose (its other substrate is ATP). It catalyzes the reaction glucose + ATP →glucose 6-phosphate + ADPInduced fit brings reactive side chains from the enzyme’s active site into alignment with the substrates, facilitating the catalytic mechanisms described earlier. Equally important, the folding of hexokinase to fit around the glucose substrate excludes water from the active site. This is essential because two molecules binding to the active site are glucose and ATP. If water was present, ATP could be hydrolyzed to ADP and phosphate. But since water is absent, the transfer of a phosphate from ATP to glucose is favored. Induced fit at least partly explains why enzymes are so large. The rest of the macromolecule may have two roles:

- It provides a framework so that the amino acids of the active site are properly positioned in relation to the substrate.

- It participates in the small but significant changes in protein shape and structure that result in induced fit.

Some enzymes require other molecules in order to operate

As large and complex as enzymes are, many of them require the presence of other, nonprotein molecules in order to function. Some of these molecular “partners” include:

Cofactors. These are inorganic ions such as copper, zinc, or iron that bind to certain enzymes and are essential to their function.

Coenzymes. These carbon-containing molecules are required for the action of one or more enzymes. Coenzymes are usually relatively small compared with the enzyme to which they temporarily bind.

Prosthetic groups. These distinctive molecular groupings are permanently bound to their enzymes. They include the heme groups that are attached to the oxygencarrying protein hemoglobin.

Coenzymes are like substrates in that they are not permanently bound to the enzyme, and must collide with the enzyme and bind to its active site. Acoenzyme can be considered a substrate because it changes chemically during the reaction and then separates from the enzyme to participate in other reactions. Coenzymes move from enzyme molecule to enzyme molecule, adding or removing chemical groups from the substrate. ATP and ADP can be considered coenzymes because they are necessary for some reactions, are changed by reactions, and bind to and detach from the enzyme. In the next chapter, we will encounter coenzymes that function in energy processing by accepting or donating electrons or hydrogen atoms. In animals, some coenzymes are produced from vitamins that must be obtained from food — they cannot be synthesized by the body. The B vitamin niacin is used to make the coenzyme NAD.

The cells of organisms carry out the basic life process of taking in and using energy.

Date: 2014-12-22; view: 1035