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

Introduction to Cell Biology
Small Molecules, Energy, and Biosynthesis
Nutrients and Cellular Energy Sources

2.3.1. Nutrient molecules are broken down in three stages to produce ATP

Proteins, Lipids, and Polysaccharides, which make up the bulk of our diet, must be broken down into smaller molecules for Cells to utilize them. This enzymatic breakdown, or Catabolism, of these molecules can be divided into three stages (Fig. 2-18). First, we will outline all of these stages in general terms, and then discuss two of them in greater detail.

In stage 1, large polymer molecules are broken down into monomeric subunits: proteins into Amino Acids, polysaccharides into sugars, and fats into Fatty acids and Cholesterol. This preliminary process, known as Digestion, occurs primarily outside cells through the action of Enzymes secreted into the lumen of the digestive tract. In stage 2, the resulting small molecules enter the cells and undergo further breakdown in the Cytoplasm. Most of the carbon and hydrogen atoms from sugars are converted into Pyruvate, which, upon entering the Cell/35.html">Mitochondria, forms the acetyl group of the reactive compound acetyl-coenzyme A (acetyl-CoA) (Fig. 2-19). Large amounts of acetyl-CoA are also produced during the Oxidation of Fatty acids. In Stage 3, the acetyl group of acetyl-CoA is completely broken down to CO₂ and H₂O. It is precisely in this final stage that the bulk of ATP is generated.

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Fig. 2-18. A simplified scheme of the three stages of catabolism leading from dietary molecules to breakdown products. This series of reactions produces ATP, which is subsequently used in biosynthetic reactions and other energy-dependent processes.

Fig. 2-19. Structure of a key metabolic intermediate—coenzyme A (acetyl-CoA). The acetyl groups generated in stage 2 of catabolism (see Fig. 2-18) are covalently linked to coenzyme A (CoA).

Through a series of coupled Chemical Reactions, more than half of the energy that, according to theoretical calculations, can be extracted from CARBOHYDRATES and fats upon their oxidation to H₂O and CO₂ is utilized to drive the energetically unfavorable reaction P_i + ADP -> ATP. Since the remainder of The energy released during oxidation is dissipated by The Cell as heat, ATP production results in an overall increase in the disorder of the Universe, which is fully consistent with The Second Law of Thermodynamics.

Through the generation of ATP, the energy initially extracted from carbohydrates and fats via oxidation is converted into a more convenient, concentrated form of chemical energy. The intracellular solution of a typical cell contains approximately 1 billion ATP molecules, the Hydrolysis of which to ADP and phosphate provides the necessary energy for numerous energetically unfavorable reactions.

2.3.2. During Glycolysis, ATP can be produced even in the absence of oxygen

The most important step in stage 2 of catabolism is glycolysis—a sequence of reactions leading to The breakdown of glucose. In glycolysis, a six-carbon glucose molecule is converted into two three-carbon pyruvate molecules. This conversion requires nine consecutive enzymatic reactions that involve The formation of a series of intermediate phosphorylated compounds (Fig. 2-20). Logically speaking, The Glycolytic Pathway can be divided into three phases: 1) in reactions 1–4, glucose is converted into the three-carbon aldehyde glyceraldehyde 3-phosphate (this conversion requires two phosphate groups, with the necessary energy provided by ATP hydrolysis); 2) in reactions 5 and 6, the aldehyde group of each glyceraldehyde 3-phosphate molecule is oxidized to a carboxyl group, and the energy released in the process is utilized to synthesize ATP from ADP and inorganic phosphate; 3) in reactions 7, 8, and 9, the two phosphate groups attached to the sugar in The first phase are transferred back to ADP, thereby generating ATP and compensating for the ATP consumed in phase 1.

Fig. 2-20. Intermediates of glycolysis. Each numbered reaction is catalyzed by a specific enzyme. In phase 4, the six-carbon sugar is split to yield two three-carbon sugars, so that following this reaction, the number of molecules doubles at each subsequent step. Reactions 5 and 6 are responsible for the net synthesis of ATP and NADH (see Fig. 2-21).

The net energy yield of glycolysis amounts to the synthesis of two ATP molecules (per glucose molecule) produced in reactions 5 and 6. Thus, these reactions are of critical importance for glycolysis. As the only reactions in the entire pathway in which a high-energy phosphate bond is formed from inorganic phosphate, they illustrate how intracellular reactions can be coupled to harness the energy released during oxidation (Fig. 2-21). The net result of these two reactions is The oxidation of the sugar aldehyde to phosphoglyceric acid, The transfer of inorganic phosphate to ADP to form a high-energy ATP bond, and the reduction of NAD⁺ to NADH (Fig. 2-22). This elegant pair of coupled reactions likely evolved at the very earliest stages of METABOLISM. Furthermore, while of paramount importance for glucose metabolism, these reactions proceed in the reverse direction during Photosynthesis, driven by NADPH and ATP generated in light-activated reactions. Consequently, they play a central role in the photosynthetic carbon-fixation process at the stage of glyceraldehyde 3-phosphate formation (see Section 7.3.4).

Fig. 2-21. Steps 5 and 6 of glycolysis: oxidation of an aldehyde to a carboxylic acid coupled with The production of ATP and NADH (see also Fig. 2-20). Step 5 begins with the formation of a covalent bond between the enzyme glyceraldehyde-3-phosphate dehydrogenase and the carbon of the aldehyde group of glyceraldehyde 3-phosphate. The carbon (in the form of a hydride ion: a proton plus two electrons) is then removed from the enzyme-bound aldehyde group of glyceraldehyde 3-phosphate and transferred to the carrier molecule NAD⁺ (see Fig. 2-22). This oxidation step yields a carbonyl group of the sugar linked to the enzyme by a high-energy bond. A phosphate ion from the solution then attacks this bond, replacing it with a high-energy sugar-phosphate bond. In these final two reactions, the enzyme couples the energetically favorable process of aldehyde oxidation with the energetically unfavorable process of high-energy bond formation. Finally, in step 6 of glycolysis, the newly formed reactive phosphate group is transferred to ADP to yield ATP, leaving a free carboxyl group on the oxidized sugar.

For most animal cells, glycolysis serves merely as a prelude to stage 3 of catabolism, since the lactic acid produced during glycolysis rapidly enters the mitochondria, where it is completely oxidized to CO₂ and H₂O. Nevertheless, in anaerobic organisms (i.e., those that do not use molecular oxygen) and Tissues capable of functioning under anaerobic conditions (such as Skeletal Muscle), glycolysis can become the primary source of cellular ATP. In these cases, rather than being degraded in the mitochondria, the pyruvate molecules remain in the Cytosol and, depending on the Organism, can be converted either into ethanol plus CO₂ (in Yeast) or into lactate (in muscle), which are subsequently eliminated from the cells. The further conversion of pyruvate in these energy-yielding reactions, known as Fermentation, is required to fully exploit the reducing power generated in step 5 of glycolysis and thereby regenerate the NAD⁺ needed for glycolysis to continue (see Section 7.4.1).

2.3.3. Oxidative catabolism yields a significantly greater amount of biologically useful energy [14]

Anaerobic ATP production from glucose via glycolysis is relatively inefficient. The end Products of Anaerobic glycolysis still retain a vast amount of chemical energy that can be released through subsequent oxidation. The evolution of oxidative catabolism (cellular Respiration) became possible only after molecular oxygen accumulated in the Earth's atmosphere As a result of photosynthesis carried out by cyanobacteria. Until that occurred, anaerobic catabolic processes were presumably the dominant form of life-support. The addition of an oxygen-requiring phase to the catabolic pathway (Stage 3 in Fig. 2-18) provides cells with a much more powerful and efficient method for extracting energy from nutrient molecules. This stage (Stage 3) begins with The Citric Acid Cycle (also called The Tricarboxylic Acid Cycle or Krebs cycle) and culminates in Oxidative Phosphorylation: both processes take place in aerobic Bacteria and the mitochondria of Eukaryotic cells.

Fig. 2-22. NADH and NAD⁺ are the most important hydrogen carriers in catabolic reactions. A. Structure of NADH and NAD⁺ molecules. NAD is the abbreviation for nicotinamide adenine dinucleotide; the name itself reflects the molecular structure, with the right-hand part (in the diagram) represented by adenosine monophosphate (AMP). The part of the NAD⁺ molecule known as the nicotinamide ring (boxed in color) can accept a hydrogen atom with an extra electron (a hydride ion, H^-), becoming reduced to NADH. In this reduced form, the nicotinamide ring is less stable because the stabilizing influence of Resonance is absent. As a result, the attached hydride ion is transferred to other molecules. B. Example of a reaction involving NAD⁺ and NADH. During Biological Oxidation of a substrate molecule, such as an alcohol, the substrate loses two hydrogen atoms. One of these is transferred as a hydride ion to NAD⁺ to form NADH, while the other is released into the solution as a proton H⁺ (see Fig. 7-18).

2.3.4. The Citric Acid cycle is the central process of metabolism [15]

The primary function of the citric acid cycle is the oxidation of the acetyl group, which enters the cycle in the form of acetyl-CoA molecules. This process is cyclical in nature because the acetyl group is not oxidized all at once, but only after it becomes covalently attached to a larger molecule, oxaloacetate, which is regenerated after each turn of the cycle. As shown in Fig. 2-23, the cycle begins with the reaction of acetyl-CoA with oxaloacetate, leading to the formation of a tricarboxylic acid molecule called citric acid (or citrate). This is followed by a series of reactions in which two of the six carbons of citrate are oxidized to CO₂, yielding a molecule of oxaloacetate—the starting reactant for a new cycle. (Since the two incoming carbon atoms added in each turn do not enter the specific part of the citrate molecule that gets oxidized to CO₂ in that same turn, several cycles must pass before their turn comes to be oxidized.) The CO₂ molecules produced in these reactions subsequently diffuse out of the mitochondria (or bacteria) and leave the cell.

Fig. 2-23. The citric acid cycle. In the mitochondria and cells of aerobic bacteria, acetyl groups derived from pyruvate undergo further oxidation. The carbon atom of the acetyl group is converted into CO₂, whereas the hydrogen atoms are transferred to the carrier molecules NAD⁺ and FAD. Additional oxygen and hydrogen atoms are incorporated into the cycle as Water molecules at the steps indicated by asterisks (*). The citric acid cycle is shown in greater detail in Fig. 7-14.

The energy released during the oxidation of C—H and C—C bonds in citrate is captured in several different ways within the citric acid cycle. In one of the cycle's reactions (succinyl-CoA to succinate), a high-energy phosphate bond is formed through a mechanism similar to the one we already encountered in glycolysis. (Although this cycle reaction produces GTP rather than ATP, all nucleoside triphosphates are energetically equivalent thanks to exchange reactions such as ADP + GTP ⇄ ATP + GDP.) The remaining energy obtained from oxidation is used to convert hydrogen carrier molecules (or hydride ions) into their reduced form; during each turn of the cycle, three NAD⁺ molecules are converted to NADH, and one flavin adenine dinucleotide (FAD) molecule becomes FADH₂. The energy carried on these carrier molecules by activated hydrogen atoms is subsequently utilized in oxidative phosphorylation—a process that requires atmospheric molecular oxygen and will be discussed in detail below.

Additional oxygen atoms required to form CO₂ from the acetyl group entering the citric acid cycle are supplied not by molecular oxygen, but by water molecules. In each cycle turn, three water molecules are split, and their oxygen atoms are incorporated into CO₂. Some of the hydrogen atoms from these water molecules bind to substrate molecules, raising them to a higher energy state, after which they are transferred (along with the hydrogen atoms of the acetyl group) to carrier molecules such as NADH.

Fig. 2-24. Generation of a transmembrane proton gradient through Electron transfer reactions. A high-energy electron (derived, for example, from the oxidation of a metabolite) is sequentially passed by carriers A, B, and C to lower energy states. In this diagram, carrier B is positioned within the membrane in such a way that as the electron passes through, it picks up an H+ ion from one side of the membrane and releases it on the other. The resulting H+ gradient serves as a form of stored energy; this energy is utilized by other mitochondrial Membrane Proteins to drive ATP synthesis (see Fig. 7-35).

In eukaryotic cells, the mitochondrion serves as the central hub toward which all Catabolic pathways converge, regardless of whether their initial substrate is sugars, fats, or proteins. This is because not only pyruvate, but also fatty acids and Certain amino acids, are transported from the cytosol into the mitochondria, where they are converted into acetyl-CoA or into one of the Intermediates of the citric acid cycle. Mitochondria also act as starting points for biosynthetic reactions, as they generate vital carbon-containing intermediates such as oxaloacetate and 2-oxoglutarate. These compounds are transported out of the mitochondrion back into the cytosol, where they serve as precursors for crucial cellular molecules, such as amino acids.

2.3.5. During Oxidative Phosphorylation, Electron Transfer to Oxygen Drives ATP Synthesis [9, 16]

Oxidative phosphorylation represents The final stage of catabolism, during which the vast majority of metabolic energy is released. In this process, NADH and FADH₂ molecules transfer electrons—originally derived from nutrient molecules—to molecular oxygen (O₂). This reaction, which is formally equivalent to the combustion of hydrogen in air to form water, releases a substantial amount of chemical energy. Part of this energy is harnessed to synthesize ATP, while the remainder is dissipated as heat.

Although the oxidation of NADH and FADH₂ ultimately results in the transfer of hydrogen to oxygen, the hydrogen is not transported in atomic form. Instead, the actual entities being transferred are the electrons of the hydrogen atom. This is because a hydrogen atom easily dissociates into its constituent parts: an electron and a proton (H⁺). The electron can then be transferred independently to a molecule that acts solely as an electron acceptor, leaving the protons behind in aqueous solution. For the same reason, when an electron alone attaches to a molecule with a strong affinity for hydrogen, a hydrogen atom is automatically formed because a proton is immediately scavenged from the solution. During oxidative phosphorylation, electrons from NADH and FADH₂ are passed along a chain of carrier molecules, with the actual form of transport (whether as a hydrogen atom or an isolated electron) depending on the specific nature of each carrier.

In eukaryotic cells, this Sequence of electron transfer reactions along the Electron Transport Chain is localized to The inner mitochondrial membrane, in which all the carrier molecules are embedded. Each step of electron transfer is accompanied by a decrease in the electrons' energy, all the way to the final destination where they are transferred to oxygen molecules. Because oxygen molecules possess the highest electron affinity, binding to oxygen places the electrons at their lowest energy level. The energy released as electrons drop to these lower energy levels is utilized to pump protons from the mitochondrial matrix to the intermembrane space, although the precise mechanism of this

process is not yet fully understood (Fig. 2-24). As a result, a transmembrane electrochemical proton gradient is established across the inner mitochondrial membrane. This gradient, in turn, drives protons to flow back down their electrochemical gradient through a specialized enzyme complex embedded in the membrane; a distinct enzyme within this complex, ATP synthase, catalyzes the addition of a phosphate group to ADP, thereby producing ATP within the mitochondrion. The newly synthesized ATP is then exported from the mitochondrion to other PARTS OF THE cell, where it powers a multitude of metabolic reactions.

The Nature of The electron transport chain and the Mechanism of ATP synthesis are discussed in detail in Chapter 7.

2.3.6. Amino Acids and NUCLEOTIDES Participate in The Nitrogen Cycle

The metabolic pathways examined in the preceding sections involved primarily Hydrocarbons. Up to this point, we have not discussed the metabolism of nitrogen or sulfur. These two elements are essential components of proteins and Nucleic Acids—the two most vital macromolecular classes of the cell, which together account for two-thirds of its dry mass. Through various transformations driven by a series of reversible cyclic processes, nitrogen and sulfur atoms cycle from one compound to another and from the tissues of various organisms into the external environment.

Molecular nitrogen is abundant in Earth's atmosphere, yet it is chemically inert. Only a small number of living species are capable of directly incorporating nitrogen into organic molecules via a process known as Nitrogen Fixation. Nitrogen fixation is carried out by specialized microorganisms and also occurs through certain geophysical phenomena, such as lightning strikes. This process is critically important for the biosphere, as life on our planet would be impossible without it. Nevertheless, in modern organisms, only a minor fraction of nitrogenous compounds originates from direct nitrogen fixation; the vast majority of organic nitrogen is continuously recycled as it passes from one organism to another. Thus, nitrogen-fixing reactions serve to replenish the global pool of available nitrogen.

Vertebrates obtain virtually all of their nitrogen from dietary proteins and nucleic acids. Within the body, these macromolecules are broken down into amino acids or nucleotides, which are subsequently used to synthesize new proteins, nucleic acids, or other molecules. Roughly half of the 20 amino acids found in proteins are classified as Essential Amino Acids (Fig. 2-25): they cannot be synthesized by the organism and must be obtained preformed from the diet. The remaining Amino acids can be synthesized from various starting Materials, including intermediates of the citric acid cycle. Essential Amino acids are produced by other living organisms—typically via lengthy, energy-demanding pathways whose metabolic routes were lost by vertebrates during evolution.

Nucleotides required for the synthesis of DNA and RNA can be produced through specialized biosynthetic pathways; there are no "essential nucleotides" that must obligatorily be supplied by the diet. All of the nitrogen atoms that make up purine and pyrimidine bases (as well as some of their carbon atoms) are derived from nitrogen-rich amino acids—glutamine, aspartic acid, and Glycine—whereas ribose and deoxyribose are derivatives of glucose.

Fig. 2-25. The nine essential amino acids that are not synthesized in human cells and must therefore be obtained from the diet.

Amino acids that are not utilized for Biosynthesis can be oxidized to yield metabolic energy. The majority of their constituent carbon and hydrogen atoms ultimately end up as CO₂ and H₂O. Meanwhile, the nitrogen atoms undergo a series of interconversions before finally being excreted from the body in the form of urea. The degradation pathways differ among individual amino acids, and the metabolism of these compounds encompasses A wide variety of enzymatic reactions.

Summary

Animal cells are thought to extract energy from food in three distinct stages. In The First stage, proteins, polysaccharides, and fats are broken down extracellularly into small molecules. In the second stage, these small molecules are degraded inside cells to yield acetyl-CoA, alongside small amounts of ATP and NADH. These reactions are unique in that they can release energy even in the absence of oxygen. In the Third Stage, acetyl-CoA molecules are broken down within the mitochondria to produce CO₂ and hydrogen atoms, which are subsequently bound to carrier molecules such as NADH. Electrons derived from these hydrogen atoms are passed along a complex chain of carriers, ultimately culminating in the reduction of molecular oxygen to form water. Driven by the energy released at various steps of electron transport, hydrogen ions (H⁺) are pumped outward from the mitochondrial matrix. The resulting transmembrane electrochemical proton gradient across the inner mitochondrial membrane provides the driving force for the Synthesis of the bulk of the cell's ATP.