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Cellular Metabolism

In order to perform all of the necessary functions for their survival, cells must be able to break complex nutrient molecules down to release the energy stored within their chemical bonds, as well as build new molecules as components for cell structure and genetic information. The sum of all of the chemical activities within the cell is called metabolism. Metabolism is composed of two parts; catabolism, or the breakdown of biological molecules, and anabolism (also called biosynthesis), which is the construction of new unique biological molecules. Catabolic reactions are exergonic, meaning that they release energy stored in the chemical bonds of molecules, while anabolic reactions are endergonic, because they store energy in the form of new chemical bonds in and between molecules. Neither catabolism nor anabolism occur as single-step events, but along multi-step metabolic pathways which either release or store energy in manageable amounts.

Enzymes

Enzymes are biological molecules which act as catalysts to speed the rate of chemical reactions within and outside of cells without being chemically altered themselves. Almost every step in each cellular
metabolic pathway is associated with at least one enzyme, and usually with more than one. No
catabolic or biosynthetic pathway would function properly without some form of enzymatic activity. Enzymes function by reducing the activation energy of chemical reactions. Activation energy is the amount of energy necessary for a chemical reaction to occur. For many biochemical reactions this is very high, thus they either do not spontaneously occur or when they do occur, release more heat than is optimal for living processes to continue. By reducing activation energy, enzymes speed these reac tions and reduce the amount of heat energy released, thus maintaining cellular temperature at a level which is optimal for other life activities. Each enzyme is specific for only one molecule, called its substrate. The enzyme binds to its substrate in a lock and key fash ion, forming an enzyme-substrate complex. While in this complex, the activation energy necessary for such chemical reactions is greatly reduced, leading to the formation of prod ucts much more quickly than would have occured spontaneously. After the catalyzed reaction has occurred, the enzyme-substrate complex breaks down, releasing the product(s), but leaving the enzyme chemically intact and capable of participating in another reaction of the same type.

Enzymes are three-dimensional structures, composed primarily of globular proteins. Each enzyme has an active site which is shaped specifically to allow the binding of only one kind of substrate. Many are also associated with ions or other organic molecules. Metal ions, called cofactors, and organic molecules, called prosthetic groups, help to complete the conformation (shape) of an enzyme, allowing it to function properly. The protein by itself is called an apoenzyme, which is nonfunctional unless bonded to its cofactor or prosthetic group. When bonded to one or both of these, it becomes a fully functional holoenzyme which can participate in enzymatic reactions. Additionally, there may be one or more allosteric sites on the molecule which can alter its shape when some substrate or product is bound or absent. In allosteric activation, the presence of a substrate bound to the allosteric site of an enzyme alters its conformation, allowing it to become chemically active by opening its active site. Conversely, in allosteric inhibition, a product of an enzyme-mediated reaction binds itself to the allosteric site of the enzyme, altering its conformation in such a way as to block its acive site, rendering it unable to bond to its substrate.

Chemical Reactions

In metabolic pathways, molecules called reactants are altered through enzyme-mediated chemical reactions to becomes one or more products. The most important of these chemical reactions to living cells include oxidation, reduction, condensation, hydrolysis, and phosphorylation. Oxidation is the loss of an electron or electrons by an atom or molecule. Reduction is the gaining of an electron by an atom or molecule. These two reactions are coupled, since when one substance loses electrons, another gains those electrons. Often in biological systems, this loss and gain of electrons is also associated with the loss and gain of hydrogen ions (H⁺), and the reactions are referred to as dehydrogenation (loss of H⁺), and hydrogenation (gain of H⁺). Oxidation- reduction (redox) reactions are important to living cells both as means of energy transfer, and in the production of new biological molecules. In biological systems, such reactions are most usually associated with organic compounds called coenzymes which are easily reduced and oxidized, serving as carriers of electrons and/or hydrogen protons that have been released from nutrient macromolecules as they are degraded. Such coenzymes include nicotinamide adenine dinucleotide (NAD), one of the most common hydrogen and electron acceptors in enzymatic biochemical pathways, nicotinamide adenine dinucleotide phosphate (NADP), and flavin adenine dinucleotide (FAD). Each of these molecules is bound to membrane and has the ability to transfer electrons and protons to sites where energy exchange takes place.

Large, complex molecules can be broken down through an enzymatic reaction called hydrolysis. In hydrolysis, a molecule of water is added between two portions of the larger molecule, which causes it to break into two parts. Conversely, large molecules can be built up from small, simple molecules called monomers through a process called condensation (also called dehydration synthesis). In condensation reactions, OH^- and H⁺ ions are removed from adjacent monomers, allowing them to form a bond between one another. The ions bond to become water, which is released. When many monomers are linked together in this way, they can form long-chain molecules called polymers. Many living organisms utilize polymers as structural components. The cell walls of plants are composed of cellulose, which is a polymer of the carbohydrate glucose. Bacterial cell walls are also composed of layers of a carbohydrate polymer called peptidoglycan, held together by amino acid linkages. Proteins are polymers of amino acids, lipids are polymers of glycerol and fatty acids or hydrocarbons, and nucleic acids are compound polymers of sugars, phosphates, and nitrogen-containing molecules called nitrogenous bases.

Phosphorylation occurs when a phosphate group (-PO₄) is added to a molecule. Often, the bond which forms between a molecule and a phosphate group retains a great deal of energy, as is the case with the primary energy storage molecule of the cell, adenosine triphosphate (ATP). ATP is composed of a primary molecule called adenosine, which is the nitrogenous base adenine bonded to the pentose (five-carbon) sugar ribose, and three phosphate groups. When the bond between adenosine and one of the phosphate groups is broken, the molecule ADP (adenosine diphosphate) is formed, and approximately 7300 calories (one calorie = the amount of energy necessary to raise 1 ml of water 1^o C) are released. This released energy can be utilized to drive cellular activities. Phosphorylation of ATP from ADP, however, does not occur spontaneously. Therefore, it is necessary to couple ATP formation with exergonic reactions during the process of cellular metabolism. If this activ ity takes place due to the release of energy during the catabolism of organic or inorganic molecules, it is referred to as substrate-level phosphorylation.

ATP can also be generated via diffusion across a cell membrane through a process called chemiosmosis. In chemiosmosis, protons released from the oxidation of nutrient macromolecules and delivered by coenzymes are forced to congregate along the outer sur face of a membrane which is not permiable to such ions. This generates a concentration gradient, resulting in the development of protonmotive force. Special enzyme channels, called ATP synthase (ATPase) molecules allow the protons to pass from the outer to the inner portion of the membrane, where they form chemical bonds with acceptors such as oxygen. The passage of these protons through ATPase drives the enzymatic activity which bonds inorganic phosphate to ADP, forming ATP.

Heterotrophic vs. Autotrophic Metabolism

Organisms which produce their own food are called autotrophs. These organisms can utilize physical factors such as sunlight (photoautotrophs) or the oxidation of inorganic compounds (chemoautotrophs) as sources of energy for the generation of ATP. In each case, carbon dioxide serves as the principle source of carbon for the biosynthesis of cell molecules and structures. Examples of photoautotrophs include the cyanobacteria, autotrophic protists such as the green, brown, red, and golden brown algaes, as well as multicellular plants. Examples of chemoautotrophs include the thermal vent bacteria such as Beggiatoa sp. and Thiomicrospira sp., nitrifying bacteria such as Nitrobacter sp., nitrogen fixing bacteria such as Rhizobium sp., and sulfur-oxidizing bacteria such as Thiobacillus sp.

Heterotrophs are organisms which must utilize metabolic pathways to oxidize organic compounds for the release of the energy necessary to generate ATP. These organisms cannot produce their own nutrients, so all carbon-containing compounds must be obtained from an outside source, either through absorption or digestion. Eubacteria, heterotrophic protists, fungi, and animals are all heterotrophs.

It is important to note that ATP generation in both heterotrophic and autotrophic organisms is associated with membrane-bound molecules, but the placement of such membranes differs. In phototrophic and chemotrophic prokaryotes such as the cyanobacteria and sulfur-using bacteria, there are internal membranes associated with chemical agents or light-activated (photosynthetic) pigments lying directly in the cytoplasm, while in eukaryotic autotrophs such as the algaes and plants, the membranes and photosynthetic pigments are housed within organelles called chloroplasts.
Heterotrophic organisms lack such pigment- associated membranes or chloroplasts. Both autotrophic and heterotrophic eukaryote cells contain speciallized organelles called mitochondria in which many of the catabolic pathways associated with nutrient oxidation take place, while in prokaryotic organisms, such reactions occur directly in the cytoplasm, in association with the plasma membrane.

Metabolic Pathways

Metabolic pathways are composed of many single-step chemical reactions which release energy in manageable amounts. All living organisms must have some original source of energy, which is generally an organic molecule, or group of organic molecules which must be disassembled or digested prior to entry into the site where such pathways take place. Some organisms, such as many bacteria and fungi begin the process of digestion outside of the body via the release of exoenzymes, which break large molecules down into forms which can cross the semi-permiable boundary of the plasma membrane. Some single-celled organisms engulf whole particles via phagocytosis or pinocytosis, thus they perform the process of digestion intracellularly, while large, multicellular organisms have evolved complex organ systems which perform the process of digestion prior to passage of nutrient molecules into the cell. Once digestion has occurred, nutrients can enter into those areas of the cell where metabolic pathways take place.

One of the most common forms of nutrients which can enter into the metabolic process is the monosaccharride glucose, which has the chemical formula C₆H₁₂O₆. This molecule is readily broken down by cells, and its elements utilized in various cellular activities. By degrading the glucose molecule via a series of enzyme-mediated reactions, the cell can make the most efficient use of the energy stored between the chemical bonds of it component parts. The process cells utilize in the breakdown of glucose is called glycolysis.

Glycolysis

Glycolysis occurs in the cytoplasm of the cell and is anaerobic, meaning that no oxygen is required for any of the chemical reactions necessary to degrade glucose. Each step is enzyme-mediated, releases energy, and results in the formation of intermediate molecules which can participate in the next step. ATP is also generated during glycolysis. However, it is necessary for the cell to degrade two molecules of ATP already present to begin the process, by breaking glucose down into two three-carbon intermediates. Thus the net gain of ATP to the cell is two less than the total number of ATP molecules generated. Additionally, during glycolysis, two molecules of NAD each accept two electrons, one hydrogen atom, and one additional hydrogen proton, which reduce them from NAD to NADH + H⁺. Ultimately, glucose is broken down into two molecules of the three-carbon molecule pyruvic acid, also called pyruvate.

Decarboxylation and Fermentation

Pyruvate can participate in a variety of different pathways, which vary according to the type of cell or organism. One of these pathways, called decarboxylation, occurs when the pyruvate is temporarily bonded by a membrane-bound molecule called coenzyme A. In decarboxylation, coenzyme A strips from pyruvate one carbon and two oxygen molecules as carbon dioxide (CO₂), which is released as waste from the cell, as well as two electrons and hydrogen protons which are collected by NAD, reducing it to NADH + H⁺. The new intermediate, acetyl coenzyme A, exists for only a short period of time, then is degraded into two molecules, the original coenzyme A which is now available to bond to another pyruvate, and a two-carbon molecule called an acetaldehyde (acetate). Acetate can either participate in the next phase of respiration, called the Krebs Tricarbolic Acid Cycle (TCA), or in a process called alcoholic fermentation.

Fermentations are chemical processes which occur an aerobically in the cytoplasm of the cell following glycolysis. In fermentation, NADH + H⁺ is oxidized back to NAD by giving the electrons and hydrogens it acquired during glycolysis to one of the organic intermediate com pounds produced as a result of this process. This does generate ATP, but the amount produced is far less than that which is the result of complete respiration. In homolactic acid fermentation, reduced NAD gives its two electrons and hydrogens to the pyruvate generated by glycolysis, which converts pyruvate to lactic acid (lactate). This form of fermentation occurs in such bacteria as the lactobacilli and streptococci, and can also occur in human cells (such as muscle tissue) if sufficient oxygen is not available for complete respiration to occur. In alcoholic fermentation, the electrons and hydrogens carried by NAD are donated to acetaldehyde, converting it to ethyl alcohol (ethanol). Fungi such as Saccharomyces cerevisiae undergo this form of fermentation, and these organisms are commonly used in the baking of bread and production of various alcoholic compounds. Some bacteria, such as various species of Lactobacillus and Leuconostoc, utilize a set of glycolytic reactions called the Entner- Douderoff pathway, wherein one molecule of pyruvate and one of acetaldehyde are produced. Since both intermediates are formed, each can be reduced by accepting the extra hydrogens and electrons carried by reduced NAD. The results of this dual reduction are one molecule each of ethanol and lactic acid per original glucose molecule, thus the process is referred to as heterolactic fermentation. Sourkraut and some types of sau sages can be produced through this form of fermentation.

Some fermentative pathways are of industrial importance, such as butyric-butylic fermentation. In this pathway, pyruvate is converted to solvent compounds such as butyric acid, butanol, isopropanol, and acetone, as well as CO₂. In propionic acid fermentation, bacteria such as Propionibacterium utilize lactate as a substrate to produce propionic acid and CO₂, which provide the flavor and holes in swiss cheese.

Still other fermentative pathways provide the basis for the differentiation of bacterial species. Bacteria such as Escherichia coli undergo mixed acid fermentation, wherein pyruvate is converted into lactic acid, acetic acid, formic acid, or combinations of these, along with molecular hydrogen (H₂) and CO₂. Other species, such as Enterobacter and Klebsiella, undergo butanediol fermentation, which results in an intermediate compound called acetyl methyl carbinol (acetoin) during the production of the neutral end product, butanediol. Two common tests used to differentiate between these species on the basis of fermentative by-products are called the methyl red (MR) and Voges- Proskauer (VP) tests. Methyl red is an acid indicator which changes color from clear to red in the presence of mixed acids, while the two indicators utilized in the VP test turn red in the presence of acetoin. E. coli produces a positive MR test, but a negative VP, while Enterobacter produces a negative MR, but a positive VP. These tests are coupled with two others, indole production and citrate fermentation, to form a combined technique called the IMViC (Indole, Methyl red Voges-proskauer Citrate) series.

The Krebs Cycle and the Electron Transport Chain

In organisms which do participate in respiration, the two-carbon acetyl group bound to coenzyme A is either released into the cytoplasm, as is the case with bacteria, or is delivered into the matrix of the mitochondrion in eukaryotic cells. It is then bonded by an enzyme to a four-carbon intermediate compound called oxaloacetic acid, to produce a new six-carbon form, citric acid. This molecule is then degraded through a series of enzyme-mediated reactions and decarboxylations back to oxaloacetic acid. As this process proceeds, hydrogens and electrons are released which reduce three NAD molecules to NADH + H⁺, a molecule of FAD to FADH₂, and enough energy is released to generate a molecule of guanosine triphosphate (GTP), which can then be used to release energy for the substrate phosphorylation of ATP. Since the original glucose molecule was split into two pyruvic acid molecules during glycolysis, each of which was decarboxylated into acetyl groups, the Krebs Cycle occurs twice for each glucose. The electrons collected by NAD and FAD molecules now reduce a series of molecules embedded in the cristae of the eukaryotic mitochondrion or the bacterial plasma membrane called cytochromes. Since cytochomes are easily reduced and oxidized, they pass the electrons along one to the next in a sequential fashion. This movement of elec trons drives the movement of hydrogen ions released by NAD and FAD across the membrane, building protonmotive force for chemiosmosis.

Ultimately, the electrons are delivered to an inorganic molecule in the cytoplasm which serves as a final electron acceptor, such as elemental oxygen in aerobic organisms, nitrate, nitrite, or sulfate in chemoautotrophic organisms. Since hydrogen ions cannot bind to cytochromes, they pass out between the inner and outer membranes of the mito chondrion or into the external environment surrounding the bacterial cell, building protonmotive force. When the hydrogen electrons pass through ATP synthase, they bond to the final inorganic electron acceptor, and in this form can be removed or reused by the cell. In the most familiar of these pathways, two hydrogens bond to elemental oxygen, forming a molecule of water (H₂O).

Cooperative Learning Activities

Complete the following paragraphs, utilizing the terms given in the list below. Some terms can be used more than once.

ATP CO₂ four enzyme electron transport chain coenzyme A NAD⁺ NADH+H⁺ P_i ten outer two water thirty-four pyruvic acid decarboxylation citric acid three FADH₂ ATPase one

As a result of glycolysis, glucose splits into __________ molecules of pyruvic acid. __________ molecules of ATP were

originally used for this process to begin, and __________ ATP molecules were produced, therefore the net gain of ATP avail

able to the cell is __________. Hydrogens and electrons released from glucose during this process were used to reduce

__________ to ________. If pyruvic acid is acted upon by __________, it is converted into acetyl coenzyme A, and a

molecule of __________ is released. The two-carbon acetyl group can then be transported to the site of the Krebs cycle,

where it is utilized through a series of __________ mediated steps to produce __________ molecules of reduced NAD,

__________ molecule of reduced FAD as __________, and one additional __________. The electrons bound by the

coenzymes are then passed to cytochromes in the __________, and the hydrogens are released to the __________ surface

of the membrane. The hydrogens then pass through __________, where they bond with oxygen to form __________. The

release of energy by electron transport, coupled with the passage of hydrogens through ATP synthase, drives the formation of

__________ new ATP molecules.

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