6: Energy, Enzymes, and Metabolism

Introduction

·         The sum total of all the chemical conversions in a cell is called metabolism.

·         The chemical buildup and breakdown of substances requires energy transformations mediated by enzymes.

Energy and Energy Conversions

·         To physicists, energy represents the capacity to do work.

·         To biochemists, energy represents the capacity for change.

·         Cells must acquire energy from their environment.

·         Cells cannot make energy; energy is neither created nor destroyed, but energy can be transformed.

·         In life, energy transformations are primarily molecular movement and changes in chemical bonds.

Energy changes are related to changes in matter

·         Two main types of energy are kinetic and potential energy. (See Figure 6.1.)

·         Potential energy is energy of state or position—it is stored energy. Potential energy is like money in the bank. It is there until you are spending it, which is when it becomes active or kinetic energy.

·         Kinetic energy is energy in action. It is doing work that alters the state or motion of matter. It is like spending your savings

·         Metabolism can be divided into two types of activities: anabolic and catabolic reactions.

·         Anabolic reactions are those that link simple molecules together to make complex ones. These are energy-storing reactions.

·         Catabolic reactions are those that break down complex molecules into simpler ones. Some of these reactions provide the energy for anabolic reactions.

The first law: Energy is neither created nor destroyed

·         Life follows the laws of physics, including the laws of thermodynamics, which apply to the whole universe or to any closed system within the universe.

·         Although living cells are open systems (they exchange matter and energy with their surroundings) they still obey this law.

·         Conservation of energy and the characteristics of energy conversions apply to both closed and open systems of matter and energy.

·         First law of thermodynamics: During any interconversion of forms of energy, the total initial energy will equal the total final energy. (See Figure 6.3a.)

The second law: Not all energy can be used, and disorder tends to increase

·         Second law of thermodynamics: When energy is being transformed, some is unavailable to do work. (See Figure 6.3b.)

·         Theoretically, a process could be 100% efficient, but certainly not greater than 100%, since this would mean more energy was available than was originally present.

·         Energy is never utilized perfectly, so the efficiency is less than 100%.

·         Total energy = usable energy + unusable energy.

·         This word equation can be more precisely expressed as: H = G + TS

·         Enthalpy is the total energy of the system, H.

·         Free energy, G, is usable energy.

·         Entropy (S) is the disorder of the system; the unusable energy is represented by entropy multiplied by the absolute temperature, T.

·         It is the usable energy that we are interested in: G = H – TS.

·         G, H, and S cannot be measured precisely.

·         However, change at a constant temperature can be measured precisely.

·         The energy changes are calculated in calories or joules.

·         The symbol D is the Greek letter delta and represents the change in a value.

·         The equation DG = DH – TDS describes the events in terms of changes in energy that occur during chemical reactions.

·         Notice there is no D in front of T since this must be constant in order to calculate other variables.

·         If DG is positive (+), then the reaction requires an input of energy. This is the case for anabolic reactions.

·         If DG is negative (–), energy is released. This is the case for catabolic reactions.

·         The efficiency of energy use is dependent on the system. Cars use the energy in gasoline to move along highways. Two different types of automobiles that weigh the same amount probably consume different amounts of fuel to go the same distance. The one that uses the least amount of fuel is most efficient.

·         Some biological systems are amazingly efficient, although never 100%. An example of an efficient use of free energy in living cells is the generation of ATP from glucose, when oxygen is used as the final electron acceptor. The details of this system are covered in Chapter 7. Although almost two dozen enzyme catalyzed reactions take place, the net capture of energy is about 68%!

·         If more disordered or random products than reactants are generated, entropy has increased, as in the hydrolysis of a protein to its amino acids.

·         When amino acids are freed from proteins, energy is released, DG is negative, and although the released energy is usually not captured in a useful form for the cell, there is an increase in entropy and a release of heat.

·         When proteins are made from amino acids, energy is used, there are fewer products, and S is negative.

·         Open versus closed systems (See Figure 6.4)

·         The universe can be simply viewed as a closed system. It has a finite amount of energy.

·         There is directionality to the universe in terms of energy. The universe is moving in the direction of greater entropy.

·         The trend toward entropy occurs because every time an energy transformation occurs, some of the energy is unusable and adds to entropy.

·         It is as if a great watch was wound up at the beginning of time and has been unwinding ever since.

·         Hydroelectric dams and living systems behave like open systems. Energy flows into and out of them. This is unlike a thermos bottle (see Figure 6.4) or the universe as a whole. However, both the dam and the cell are part of the universe and contribute to the increase in universal entropy. Not all the energy of either is directed perfectly to do work. With each energy transformation, there is an increase in entropy.

·         Earth acts as an open system, which receives its energy from the environment, mostly the sun. Living things use this energy to create order (reduce entropy) locally, but the overall entropy of the solar system invariably increases.

Chemical reactions release or take up energy

·         Chemical reactions either consume or release energy.

·         Anabolic reactions may make single products from many smaller units; such reactions consume energy.

·         Catabolic reactions may reduce an organized substance, such as a glucose molecule, into smaller more randomly distributed substances, such as carbon dioxide and water; such reactions release energy.

·         There is a direct relationship between the amount of energy released by a reaction (–DG) or the amount taken up (+DG) and the tendency of a reaction to run to completion without an input of energy.

·         A spontaneous reaction goes more than halfway to completion without input of energy. (See Figure 6.5a.)

·         Nonspontaneous reactions proceed that far only with an input of energy. (See Figure 6.5b.)

·         Spontaneous reactions are called exergonic and have negative DG values (they release energy).

·         Nonspontaneous reactions are called endergonic and have positive DG values. (they consume energy).

·         If under certain conditions A ® B is spontaneous (and exergonic), then B ® A must be nonspontaneous (and endergonic).

·         Making protein is endergonic; hydrolyzing protein is exergonic.

·         All reactions in principle may be reversible. (A « B)

·         Adding more A speeds up the forward reaction, A ® B; adding more B speeds up the reverse reaction, B ® A.

·         At some relative concentration of A and B, forward and reverse reactions take place at the same rate.

·         At this point, no further net change occurs. However, reactions of individual molecules continue.

·         This point is called chemical equilibrium. (See Figure 6.6.)

Chemical equilibrium and free energy are related

·         An example of equilibrium is when glucose 1-phosphate is converted in the cell to glucose 6-phosphate.

·         At pH 7 and 25oC, the equilibrium of product-to-reactant is 19:1.

·         The forward reaction has gone 95% to completion.

·         The farther a reaction goes toward completion in order to reach equilibrium, the greater the amount of free energy released.

ATP: Transferring Energy in Cells

·         ATP is adenosine triphosphate. Figure 6.8 shows the chemical diagram of ATP.

·         All living cells use ATP for capture, transfer, and storage of energy.

·         ATP may be thought of as the energy “currency ” of the cell—“the coin of the realm.”

·         ATP is not an unusual molecule and may be converted to other uses, such as building blocks for DNA and RNA

ATP hydrolysis releases energy

·         ATP consists of the nitrogenous base adenine bonded to ribose. Carbon 5 of the ribose has three phosphate groups attached.

·         ATP can hydrolyze to yield ADP and an inorganic phosphate ion (Pi, short for HPO42-).

·         ATP + H2O ® ADP + Pi + free energy. The change in free energy (DG) is –12 kcal/mol at a living cell’s typical temperature and pH.

·         The equilibrium is far to the right of the equation, toward ADP production; there are 10 ´ 106 ADP molecules to each remaining ATP.

·         Making ATP from ADP involves overcoming repulsive negative charges on the phosphates to be joined. The energy to do this is stored in glucose or other fuel molecules and released in the endergonic process. (See Figure 6.9.)

ATP couples exergonic and endergonic reactions

·         Where does the energy come from to make ATP?

·         ADP + Pi + free energy ® ATP + H2O

·         Exergonic reactions are coupled to the endergonic reaction of making ATP. The free energy is captured in ATP.

·         The energy to make ATP comes from the energy released from fuel molecules such as glucose.

·         ATP shuttles energy from exergonic reactions to endergonic reactions.

·         Figure 6.10 provides a sketch of a coupled reaction that uses ATP to provide energy for the production of glutamine from an ammonium ion and glutamate.

·         The reaction actually involves two steps; in the first reaction, glutamate gets a phosphate group from ATP (glutamate is phosphorylated) and becomes a compound called glutamyl phosphate.

·         The second reaction involves hydrolysis of glutamyl phosphate. This provides the free energy to drive the reaction with an ammonium ion to form glutamine.

·         The example above is very typical of how ATP is used in synthesis reactions.

·         Each cell requires millions of molecules of ATP per second to drive its biochemical machinery.

·         Each ATP molecule undergoes about 10,000 cycles of synthesis and hydrolysis every day.

Enzymes: Biological Catalysts

·         A catalyst is any substance that speeds up a chemical reaction without itself being used up. Living cells use biological catalysts to increase rates of chemical reactions.

·         Most biological catalysts are proteins called enzymes. Certain RNA molecules are catalysts and are called ribozymes.

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

·         It is easy to predict the direction a spontaneous reaction will go, but not the likelihood or rate of the reaction.

·         The direction of the spontaneous reaction of wood with oxygen and the levels of end products in our environment allow us to predict that forests will burn. However, it might be a few years or a few hundred years before this burning occurs.

·         An initial investment of energy must be made, like the energy from a lightning strike.

·         When the energy from the combustion of wood is released, some of the energy is invested in the unburned molecules of wood to perpetuate the fire.

·         The energy that must be invested to initiate a reaction is called its activation energy. The Instructor’s Resource CD-ROM includes a photograph of a balanced rock as an illustration of this concept; the activation energy of a reaction corresponds to the energy required to push the rock off its perch.

·         All reactions have activation energy requirements, even extremely exergonic ones.

·         Different reactions have different activation energy requirements.

·         Activation energy is the energy needed to put molecules into a transition state. The molecules must become unstable transition-state species.

·         Transition-state species have higher free energy than either reactants or products. (See Figure 6.11.)

·         Adding enough heat to increase the average kinetic energy of the molecules is often how exergonic reactions are initiated, as in the case of  the lightning strike.

·         Adding heat is not always an appropriate way for biological systems to drive reactions. Enzymes, acting as biological catalysts, solve this problem.

·         Catalysts do not cause a reaction to take place that could not take place eventually without it.

·         Catalysts substantially lower the required energy of activation.

Enzymes bind specific reactant molecules

·         Almost all enzymes are proteins, made of simple amino acids.

·         Although made of amino acids, some enzymes have important contributing molecules that participate in the catalysis, such as some types of vitamins and metal ions.

·         Enzymes bind specific reactant molecules called substrates.

·         Substrates bind to a particular site on the enzyme surface called the active site, where catalysis takes place. (See Figure 6.13).

·         Enzymes are highly specific. They bind specific substrates and catalyze particular reactions under certain conditions.

·         An enzyme that catalyzes a certain reaction in one species might differ in amino acid composition from the corresponding enzyme in another species, especially if they live at different temperatures and/or ionic environments.

·         The specificity for substrates comes from the 3D shape of the enzyme.

·         The amino acid sequence, temperature, and other solution or environmental conditions determine the 3D shape and structure of the active site.

·         The name of the enzyme relates to its function. Most, but not all enzyme names end in the suffix “ase.”

·         RNA polymerase catalyzes formation of RNA but not DNA.

·         RNA nuclease hydrolyzes RNA polymers.

·         Hexokinase accelerates phosphorylation of hexose. (All kinases add phosphates. All phosphatases remove phosphates.)

·         Binding a substrate to the active site produces an enzyme–substrate complex.

·         Hydrogen bonding, ionic attraction, or covalent bonding acting individually or together hold these complexes together.

·         The enzyme–substrate (ES) complex generates the product (P) and free enzyme (E): E + S ® ES ® E + P. (See Figure 6.13.)

Enzymes lower the activation energy barrier but do not affect equilibrium

·         Enzymes and other catalysts lower activation energy requirements but do not affect equilibrium. (See Figure 6.14.)

·         The equilibrium is determined by the nature of the reaction and the conditions; it is the relative concentration of reactants and products, when no further net change is observed.

·         Enzymes accelerate both the forward and reverse reactions, reducing the time it takes to reach equilibrium.

·         Enzyme action does not alter the difference in free energy between the reactants and the products, only the rate of the reaction.

·         An example is lactate dehydrogenase. This enzyme catalyzes both the conversion of pyruvate to lactate, and of lactate to pyruvate.

·         Which is the substrate? Both pyruvate and lactate can be the substrate or product, depending on the relative concentration of each. If lactate concentrations are high, then it is the substrate. If pyruvate concentrations are high, than it is the substrate.

·         Enzymes can have a profound effect on rates toward equilibrium. Reactions that might take years to happen can occur in a fraction of a second.

What are the chemical events at active sites of enzymes?

·         At the active sites enzymes and substrates interact by breaking old bonds and forming new ones. Enzymes catalyze reactions using one or more of the following mechanisms: orienting substrates, adding charges to substrates, or inducing strain in the substrates.

·         Enzymes orient substrates.

·         While free in solution, substrates tumble and collide.

·         The probability for the collision at the angle necessary to change chemical interactions is low.

·         When bound to enzymes, two substrates can be oriented such that a reaction is more likely.

·         Enzymes add charges to substrates.

·         The R groups (side chains) of an enzyme’s amino acids may directly participate in making substrates more chemically reactive.

·         Some enzymes work by what is called acid-base catalysis.

·         Acidic or basic side chains of amino acids form the active site and transfer H+ to or from the substrate, destabilizing a covalent bond in a substrate.

·         Some enzymes use covalent catalysis. A functional group side chain forms a temporary covalent bond with the substrate.

·         Some have metal ion cofactors, which involve the gain or loss of electrons, called oxygen-reduction (redox) reactions.

·         Some enzymes induce strain in the substrate.

·         For example, the carbohydrate substrate for the enzyme lysozyme enters the active site in a flat-ringed "chair" shape.

·         The active site causes it to flatten out into a "sofa" shape.

·         The stretching of the bonds decreases their stability, making them more reactive to water.

Substrate concentration affects reaction rate

·         The rate of an uncatalyzed reaction is directly proportional to the concentration of reactants. The higher the substrate concentration, the more collisions and reactions per unit of time.

·         This is true to a point with catalyzed reactions. At some point the enzyme will have all active sites occupied (saturated).

·         Saturating an enzyme makes it possible to determine how many molecules are converted per unit time. (See Figure 6.16.)

·         The turnover number ranges from 1 molecule every 2 seconds for lysozyme, to 40 million per second for the liver enzyme catalase.

Molecular Structure Determines Enzyme Function

·         Most enzymes are much larger than their substrate.

·         The active site of most enzymes is only a small region of the whole protein.

The active site is specific to the substrate

·         The specificity of an enzyme for a particular substrate depends on a precise interlock.

·         In 1894, Emil Fischer compared the fit to that of a lock and key.

·         In 1965, using X-ray crystallography, David Phillips observed a pocket in the enzyme lysozyme that neatly fit its substrate. (See Figure 6.17.)

An enzyme changes shape when it binds a substrate

·         Studies on enzyme inhibitors showed that purposely modified substrates could bind an enzyme without being affected by the enzyme.

·         Some enzyme inhibitors were larger than the normal substrate, yet could occupy the binding site.

·         The conclusion is that some enzymes are flexible; the active site can change shape.

·         This is called induced fit.

·         Enzymes are much larger than their substrates or reactants.

·         Part of the larger size might be what allows induced fit. (See Figure 6.18.)

·         Some regions of an enzyme tolerate little change without the enzyme losing its activity. This is the case within the active site.

·         Some enzymes can tolerate small changes in amino acid composition outside the active site and provide a framework that enhances induced fit.

To operate, some enzymes require added molecules

·         Cofactors are inorganic ions such as copper, zinc, and iron that bind temporarily to certain enzymes and are essential to their function

·         Coenzymes are carbon-containing molecules required for the action of one or more enzymes. (See Figure 6.19.) Some coenzymes are vitamins.

·         Prosthetic groups are permanently bound to enzymes. They include the heme groups (iron-containing organic molecules) that are attached to hemoglobin.

·         Table 6.1 is a list of examples of cofactors, coenzymes, and prosthetic groups.

·         Coenzymes must react with an enzyme, separate, and then participate in other reactions.

·         ATP and ADP are coenzymes. They are also substrates of the reactions.

Metabolism and the Regulation of Enzymes

·         In general, enzyme activity must be regulated. There is a specific time for such things as RNA synthesis and glucose breakdown.

·         This regulation contributes to cell homeostasis, the maintenance of a stable internal state best suited to cell survival.

Metabolism is organized into pathways

·         Metabolism is organized into sequences of enzyme-catalyzed chemical reactions called pathways.

·         A simple diagram of a biochemical pathway is as follows:

enzyme
 

enzyme
 

enzyme
 
A                      B                   C                    D

 

·         Each step A to B to C to D occurs appropriately because of enzymes. For example, one enzyme converts A to B; a second enzyme converts B to C.

·         Some metabolic pathways are anabolic and synthesize the building blocks of macromolecules.

·         Some are catabolic and break down macromolecules and fuel molecules.

·         In life, all reactions, taken together, are net exergonic, or energy releasing.

·         All anabolic reactions are coupled to catabolic ones in a dynamic balance.

·         The life we are most familiar with uses one important reaction to get the energy for the rest—the capture of a photon of light into a chemical bond.

·         Some other less familiar life forms use the energy of methane or reduced inorganic substances as the starting energy source for all other reactions.

Enzyme activity is subject to regulation

·         Enzymes can have their activity inhibited by natural and artificial binders.

·         Irreversible inhibition occurs when the inhibitor destroys the enzyme’s ability to interact with its normal substrate(s).

·         Diisopropylphosphorofluoridate, for example, reacts with the OH group of the serine residues found in active sites, eliminating the activity of the enzyme.

·         Sarin, a related compound, is the nerve gas that was released in the Tokyo subway, causing death and illness.

·         Irreversible inhibitors are usually artificial, human-made compounds. Nature uses reversible inhibition to regulate metabolism. (See Figure 6.21.)

·         When an inhibitor binds reversibly to an enzyme’s active site, it competes with the substrate for the binding site and is called a competitive inhibitor.

·         When an inhibitor binds reversibly to a site away from the active site, it is called a noncompetitive inhibitor. Noncompetitive inhibitors act by changing the shape of the enzyme in such a way that the active site no longer binds the substrate.

Allosteric enzymes have interacting subunits

·         Allosteric enzymes may have interacting subunits that change in shape and function and modulate their catalytic activity. Allo means different, and steric means shape.

·         Allosteric enzymes are controlled by effector molecules.

·         Effector molecules bind to an allosteric site, which is separate from the active site.

·         This binding changes the structure and function of the enzyme.

·         Depending on the particular enzyme, the binding may enhance or diminish reactions at the active site.

·         Some allosteric enzymes have multiple active sites.

·         When one binding site is occupied, it changes the other(s) so they bind additional substrate molecules more readily.

·         This changes the shape of the reaction rate versus concentration curve compared to non-allosteric enzymes. (See Figure 6.22.)

·         The advantage to the system is that the enzyme's catalytic rate becomes concentration sensitive and responsive.

·         Allosteric enzymes usually have more than one type of subunit.

·         A catalytic subunit has an active site that binds the enzyme's substrate.

·         A regulatory subunit has one or more allosteric sites that bind specific effector molecules.

·         An allosteric enzyme exists in an active or inactive form, similar to having an on/off switch.

·         In the active state, the active sites on the catalytic subunits can bind substrate.

·         In the inactive state, the allosteric sites on the regulatory subunits can accept inhibitor.

·         See Figure 6.23 for more information on the kind of subunits and nature of regulatory effects.

·         Allosteric effects regulate metabolism.

·         Metabolic pathways typically involve a starting material, intermediates, and a useful end-product.

·         The first step in the pathway is called the start up or commitment step.

·         Once this step occurs, other enzyme-catalyzed reactions follow until the product of the series builds up.

·         One way to control the whole pathway is to have the end-product inhibit the first step in the pathway, the commitment step.

·         This is called end-product inhibition. (See Figure 6.24.)

Enzymes and their environment

·         pH and temperature and can affect enzyme activity.

·         Each enzyme is most active at a certain pH and temperature. (See Figures 6.25 and 6.26.)

·         Some enzymes are tolerant to a wide range of pH and temperatures, while other enzymes are very sensitive.

·         pH can influence the charges of carboxyl groups in neutral or basic solutions and amino groups in neutral or acidic solutions.

·         In general, an increase in temperature (to a point) increases the rate of an enzyme-catalyzed reaction. All enzymes, as specific entities, show an optimal temperature for activity.

·         Temperature can negatively influence shape by breaking hydrogen bonds and by interfering with ionic interactions and hydrophobic interactions. If heat destroys the enzyme, the enzyme is called denatured.

·         Some organisms that live at different temperatures generate different forms of an enzyme, called isozymes.

The Instructor’s Resource CD-ROM includes photographs illustrating activation energy and the flow of energy from producers to consumers.