7: Cellular Pathways That Harvest Chemical Energy

Introduction

· Several principles govern metabolic pathways in cells.

· Complex chemical reactions occur in many steps connected in a pathway.

· A specific enzyme catalyzes each reaction.

· Metabolic pathways are similar in all organisms.

· Despite the use of many specific enzymes in catalyzing reactions, metabolic pathways are regulated by a few enzymes utilizing allosteric controls. (See Figure 7.21).

Obtaining Energy and Electrons from Glucose

· The sugar glucose (C₆H₁₂O₆) is the most common form of energy molecule.

· Fats, a variety of sugars, and proteins can be consumed for energy. However, all organisms use glucose as a primary fuel molecule.

· Almost all foods that are used for energy are converted to glucose or to an intermediate compound.

Cells trap free energy while metabolizing glucose

· When burned in a flame, glucose releases heat, carbon dioxide, and water.

· C₆H₁₂O₆ + 6 O₂ ® 6 CO₂ + 6 H₂O + energy (heat and light).

· The same equation applies for the biological, metabolic use of glucose. This process, however, has many steps and is carefully controlled.

· About half of the energy is collected in ATP.

· DG for the complete conversion of glucose is –686 kcal/mol.

· The reaction is therefore highly exergonic, and it drives the endergonic formation of ATP.

· Some kinds of cells metabolize glucose incompletely, and others completely.

· The incomplete breakdown is called fermentation and is anaerobic (no oxygen required).

· Fermentation has steps that are common to all organisms.

· Complete capture of the energy in the hydrocarbon bonds of glucose requires oxygen.

· The two main metabolic processes for complete use of glucose are glycolysis and cellular respiration.

· Glycolysis is a series of reactions that produce some usable energy and two molecules of a three-carbon sugar called pyruvate.

· Cellular respiration occurs in aerobic (oxygen-containing) environments. Pyruvate is converted to CO₂ and H₂O. The energy from hydrocarbon bonds is used to make ATP molecules.

· If cellular respiration does not occur, then fermentation does.

· Fermentation occurs when the environment is anaerobic (lacking gaseous oxygen).

· It also happens whenever a eukaryotic cell has no mitochondria, which is rare. Cellular respiration in eukaryotic cells occurs in the mitochondria.

· In fermentation, lactate or ethanol and CO₂ are produced, but far less energy is extracted from glucose. (See Figure 7.1.)

Redox reactions transfer electrons and energy

· Redox reactions transfer the energy of electrons.

· A gain of one or more electrons or hydrogen atoms is called reduction.

· The loss of one or more electrons or hydrogen atoms is called oxidation.

· Whenever one material is reduced, another is oxidized. (See Figure 7.2).

· When deciding whether an organic compound is being oxidized or reduced, look at the carbons in the compound and note the changes in the number of bonds they have with hydrogen or oxygen.

· If a carbon gains a bond with hydrogen, the compound is reduced.

· If a carbon loses a bond with hydrogen, the compound is oxidized.

· Reaction 1 of the above diagram is oxidation because the changed carbon loses a bond with hydrogen.

· Reaction 2 is also oxidation because the central carbon in the reactant loses a hydrogen bond (even though the compound as a whole still has the same amount of hydrogen).

· An oxidizing agent accepts an electron or a hydrogen atom.

· A reducing agent donates an electron or a hydrogen atom.

· During both the process of burning glucose and the metabolism of glucose, glucose is the reducing agent (and is oxidized), while oxygen is the oxidizing agent (and itself is reduced).

· The overall DG of a biologically related redox reaction is negative.

The coenzyme NAD is a key electron carrier in redox reactions

· The molecule NAD is an essential electron carrier in cellular redox reactions.

· NAD is short for nicotinamide adenine dinucleotide.

· NAD exists in an oxidized form, NAD⁺, and a reduced form, NADH + H⁺.

· NAD⁺ + 2 H ® NADH + H⁺ is the reduction reaction, and requires an input of energy.

· Oxygen reacts with NADH + H⁺:

NADH + H⁺ + 1/2 O₂ ® NAD⁺ + H₂O

· The DG of this oxidation reaction is -52.4 kcal/mol. (For comparison, the DG of the ATP to ADP reaction is –12 kcal/mol.)

· Think of NAD as a packaging agent for free energy.

· Another electron carrier is FAD (flavin adenine dinucleotide).

An Overview: Releasing Energy from Glucose

· The energy in glucose is contained in the reduced hydrocarbon bonds.

· To capture this energy, glucose must be oxidized.

· With oxygen and a means to use it, four major pathways operate:

· Glycolysis splits glucose into two pyruvate molecules.

· Pyruvate oxidation results in acetate, a compound requiring further metabolism.

· The citric acid cycle uses the product of pyruvate oxidation.

· The respiratory chain uses the products of the citric acid cycle and of glycolysis to produce more ATP.

· When no oxygen is available, or the cells are types that are unable to use it, only glycolysis occurs. A fermentation reaction converts the pyruvate molecules. (See Figure 7.5.)

· The location of these processes in cells is shown in Table 7.1

Glycolysis: From Glucose to Pyruvate

The energy-investing reactions of glycolysis require ATP

· Glycolysis moves glucose to pyruvate.

· To get energy out of glucose, energy first must be invested.

· In separate reactions, two ATP molecules are used to make modifications to glucose.

· Phosphates from each ATP are added to the carbon 6 and carbon 1 of the glucose molecule.

· See Figures 7.6 and 7.7 for the details of this part of glycolysis.

· Note that the enzyme in the first reaction is hexokinase. (All kinases add phosphates.)

· The enzyme aldolase splits the molecule into two three-carbon molecules that then both become glyceraldehyde 3-phosphate (G3P).

· To this point, no energy has been yielded.

The energy-harvesting reactions of glycolysis yield ATP and NADH + H⁺

· See Figure 7.7 for the energy-yielding reactions of glycolysis. A reaction catalyzed by the enzyme triose phosphate dehydrogenase releases a lot of free energy, more than 100 kcal per mole of glucose.

· The energy is stored rather than being lost by heat and is used to make two molecules of NADH + H⁺, one for each of the two glyceraldehyde molecules made from the one glucose molecule.

· Two other reactions each yield one ATP per glyceraldehyde or four ATP molecules per glucose molecule. This part of the pathway is called substrate-level phosphorylation.

· The final product is two of the three-carbon molecule pyruvate, which still has some reduced carbon bonds.

· Glycolysis might be followed by fermentation.

· In fermentation, the yield of captured energy for each glucose molecule is just two ATP molecules per glucose.

Pyruvate Oxidation

· Pyruvate is oxidized to acetate. This forms the link between glycolysis and cellular respiration.

· Pyruvate oxidation occurs in a multistep reaction catalyzed by an enormous enzyme complex that is attached to the inner mitochondrial membrane.

· The acetyl group is added to coenzyme A to form acetyl CoA. A molecule of NADH + H⁺ is generated during this reaction, capturing approximately 50kcal/mole. (See Figure 7.8.)

· As two pyruvate molecules are generated from each glucose molecule during glycolysis, two NADH + H⁺ per glucose are generated during this stage of glucose oxidation.

· This reaction requires a complex series of steps completed by the pyruvate dehydrogenase complex.

· This enzyme complex is extremely large (4.6 million daltons) and consists of 72 polypeptide chains of three different types.

The Citric Acid Cycle

· Pyruvate oxidation prepares the hydrocarbons for the citric acid cycle.

· Acetyl CoA is the starting point for the citric acid cycle.

· The pathway has eight reactions and occurs in the mitochondria.

· The reaction completely oxidizes the carbons of the acetate.

The citric acid cycle produces two CO₂ molecules and reduced carriers

· The products of the citric acid cycle include two molecules of CO₂.

· Free energy is captured by NAD⁺ and FAD⁺ to generate the reduced form of each; some free energy is also captured in the synthesis of ATP from ADP.

· The cycle begins when the two carbons from the acetate are added to oxaloacetate, a four-carbon molecule, to generate citrate, a six-carbon molecule.

· A series of reactions oxidize two carbons from the citrate. With some enzyme-catalyzed molecular rearrangements, oxaloacetate is formed, and this can be used for the next cycle.

· For each turn of the cycle, three molecules of NADH + H⁺, one molecule of ATP, and one molecule of FADH₂ are generated. (The FADH₂ is another reducing agent similar to NADH.)

· As two acetate molecules are contributed from each glucose, twice this number of high-energy molecules are generated per glucose. See Figure 7.10 for a complete accounting.

· Some of the products of the citric acid cycle are starting substrates for other necessary molecules like nucleotides and amino acids.

· This cycle is maintained in a steady state as the concentrations of molecules do not change much.

· This complex of reactions is far more efficient at tapping off energy than a single reaction would be.

The Respiratory Chain: Electrons, Proton Pumping, and ATP

· The respiratory chain, the final component of glucose oxidation, is needed to make use of the generated reducing agents and the energy they possess.

· By relieving them of their hydrogens (H⁺ + e^-), respiration also replenishes the supply of the oxidized forms: FAD and NAD⁺.

· Electrons pass along a series of membrane-associated electron carriers called the respiratory chain.

· The flow of electrons by way of a series of redox reactions causes the active transport of protons across the inner mitochondrial membrane and into the intermembrane space, creating a concentration gradient.

· The protons then diffuse through proton channels down the concentration and electrical gradient back into the matrix of the mitochondria, creating ATP in the process.

· The entire process of ATP synthesis by electron transport through the respiratory chain is called oxidative phosphorylation.

The respiratory chain transports electrons and releases energy

· ATP is generated because the protons move through a transmembrane protein called ATP synthase. This enzyme makes ATP from ADP and P_i.

· The respiratory chain transports electrons and releases energy.

· Protein complexes are bound to the inner mitochondrial membrane.

· The inner membrane is highly folded to increase surface area.

· Figure 7.11 shows a functional diagram of the mitochondrial inner membrane.

· NADH + H⁺ passes its hydrogen atoms to the NADH-Q reductase protein complex. The NADH-Q reductase passes the hydrogens on to ubiquinone (Q) forming QH₂.

· The QH₂ passes electrons to cytochrome c reductase, which in turn passes them to cytochrome c. Next to receive them is cytochrome c oxidase. The cytochrome oxidase passes them on to oxygen.

· Reduced oxygen unites with two hydrogens to form water.

· See Figure 7.12 for a complete review of the respiratory chain.

· FADH₂ can also enter the chain through succinate-Q reductase.

· This complex series of reactions actually tames the energy release by only allowing small, manageable energy packets to form.

Active proton transport is followed by diffusion coupled to ATP synthesis

· As electrons pass through the respiratory chain, protons are pumped into the intermembrane space against a concentration and electrical gradient (See Figure 7.13).

· The potential energy generated is called the proton-motive force.

· The chemiosmotic mechanism couples electron transport to ATP synthesis. It has three parts:

· NADH or FADH₂ (reduced forms) yield energy upon oxidation.

· The energy is used to pump protons into the intermembrane space, contributing to the proton-motive force.

· The potential energy from the proton-motive force is harnessed by ATP synthase to synthesize ATP from ADP.

· Synthesis of ATP from ADP is reversible: Excess ATP can be hydrolyzed to ADP + P_i + free energy.

· Why is this reaction driven toward ATP synthesis and not hydrolysis in mitochondria?

· The synthesized ATP is transported out of the mitochondrial matrix as quickly as it is made.

· The proton gradient is kept high by the pumping of the electron transport chain because of the availability of fuel molecules such as glucose.

· In mammals, ATP synthase is a multi-protein machine with 16 different polypeptides.

· Two functional parts of the machine are a membrane channel for H⁺ and a lollipop-shaped portion that protrudes into the matrix.

· The movement of a proton through the channel causes the physical rotation of the core of the enzyme.

· This pushes the ADP and P_i so close together that they bond.

· Figure 7.14 shows the initial experiments that proved the chemiosmotic mechanism.

· Proton diffusion can be used to create only heat when uncoupled from ATP production.

· If a simple proton channel without ATP synthase is added, then the potential energy of the proton-motive force is transferred to heat energy.

· A protein called thermogenin does just this to generate heat in newborn humans and hibernating animals.

Fermentation: ATP from Glucose, without O₂

· When there is an insufficient supply of oxygen, a cell cannot reoxidize cytochrome c.

· Then QH₂ cannot be oxidized back to Q, and soon all the Q is reduced.

· This continues until the entire respiratory chain is reduced.

· NAD and FAD are not generated from their reduced form.

· Pyruvate oxidation stops, due to a lack of NAD.

· Likewise, the citric acid cycle stops, and if the cell has no other way to obtain energy, it dies.

· Thus, oxygen at the end of the respiratory chain reaction acts as the ultimate electron acceptor.

· Some cells under anaerobic conditions continue glycolysis and produce a limited amount of ATP by fermentation.

Some fermenting cells produce lactic acid and others produce alcohol

· In the absence of oxygen or in eukaryotic cells that lack mitochondria, pyruvate builds up as the end product of glycolysis, and NAD⁺ supplies dwindle. Since the supply of NAD⁺ is limited, NADH + H⁺ must be converted back to NAD⁺.

· The NADH + H⁺ concentration builds up and an enzyme reduces pyruvate using NADH + H⁺ to do so.

· In lactic acid fermentation, an enzyme, lactate dehydrogenase, uses the reducing power of NADH + H⁺ to convert pyruvate into lactate, and NAD⁺ is replenished in the process. (See Figure 7.15.)

· Lactic acid fermentation occurs in some microorganisms—even in our muscle cells when they are starved for oxygen.

· Alcohol fermentation involves the use of two enzymes to metabolize pyruvate. (see Figure 7.16.)

· First carbon dioxide is removed from pyruvate leaving acetaldehyde.

· Then NADH + H⁺ is used by the second enzyme to convert the acetaldehyde to ethanol.

· Without oxygen, the yield of energy is low.

· NADH + H⁺ is simply consumed.

· Four ATP molecules are generated per glucose molecule, but two had to be invested.

· The net yield therefore is just two ATP per glucose molecule.

Contrasting Energy Yields

· A total net of 38 ATP molecules can be generated from each glucose molecule in glycolysis followed by complete aerobic respiration.

· Some animal cells' inner mitochondrial membranes are impermeable to NADH. To get into the matrix requires the energy of one ATP for each of the two NADH produced per molecule of glucose. This reduces the yield to 36 ATP molecules. (See Figure 7.17.)

· In contrast, fermentation has a net yield of only 2 ATP molecules from each glucose molecule. (See Figures 7.15 and 7.16.)

· The end products of fermentation (such as lactic acid and ethanol) contain much more unused energy than the end products of aerobic respiration.

· In aerobic respiration, each NADH + H⁺ generates three ATP molecules, and each FADH₂ generates two ATP molecules when consumed in the electron transport chain.

· Coupled with glycolysis, aerobic respiration captures 63 percent of the energy stored in glucose; fermentation captures only 3.5 percent. Aerobic respiration is 18 times more efficient at harvesting energy from glucose.

Metabolic Pathways

· Glucose utilization pathways can yield more than just energy. They are interchanges for diverse biochemical traffic.

· Intermediate chemicals are generated that are substrates for the synthesis of lipids, amino acids, nucleic acids, and other biological molecules. (See Figure 7.18).

Catabolism and anabolism involve interconversions using carbon skeletons

· Catabolic and anabolic pathways intersect around the energy-yielding pathways.

· Catabolic uses of molecules include the following:

· Polysaccharides are hydrolyzed into sugars, which pass on to glycolysis.

· Lipids are converted to fatty acids, which become acetate (and then acetyl CoA), and glycerol, which is converted to dihydroxyacetone phosphate, an intermediate in glycolysis.

· Proteins are hydrolyzed into amino acids, which feed into glycolysis or the citric acid cycle.

· Anabolic interconversions include the following:

· Gluconeogenesis is the process by which intermediates of glycolysis and the citric acid cycle are used to form glucose.

· Acetyl CoA can form fatty acids. Amino acids can be polymerized into proteins.

· Common fatty acids have even numbers of carbons because they are formed by adding two-carbon acetyl CoA “units.”

· The citric acid cycle intermediate a-ketoglutarate is the starting point for the synthesis of purines. Oxaloacetate is a starting point for pyrimidines. (See Figure 7.19.)

Catabolism and anabolism are integrated

· The levels of the products and substrates of energy metabolism are remarkably constant.

· Cells regulate the enzymes of catabolism and anabolism to maintain a balance.

· What happens if inadequate fuel molecules are available?

· Polysaccharides are an intermediate storage form of energy. A typical person has about one day's worth of energy stored as the carbohydrate glycogen.

· A typical person has about a week of needed amino acids stored as protein, and over a month's energy stored as fats.

· Fats are compact energy storage molecules because they exclude water and are particularly hydrocarbon rich.

· If food is withheld, glycogen is used up first, then fats.

· Fats cannot cross the blood–brain barrier. To supply glucose to the brain, glucose must be synthesized by gluconeogenesis. This requires the use of amino acids.

· Therefore, proteins must be broken down.

· After fats are depleted, proteins alone provide energy.

· At this point important disease-fighting proteins (antibodies) are consumed, and the likeliness of severe illness increases.

Regulating Energy Pathways

· Metabolic pathways work together to provide cell homeostasis.

· Positive and negative feedback controls whether a molecule of glucose is used in anabolic or catabolic pathways.

Allostery regulates metabolism

· Figures 7.20 and 7.21 diagram allosteric regulation. The amount and balance of products a cell has is regulated tightly.

· This balance is achieved via allosteric regulation of enzyme activities. Control points use both positive and negative feedback mechanisms.

· The main control point in glycolysis is the enzyme phosphofructokinase.

· This enzyme is inhibited by ATP and activated by ADP and AMP.

· When ATP is low, phosphofructokinase is active; when ATP is high, it is inactive.

· The main control point of the citric acid cycle is the enzyme isocitrate dehydrogenase.

· This enzyme converts isocitrate to a-ketoglutarate.

· NADH and ATP are inhibitors of this allosteric enzyme.

· NAD and ADP are activators of it.

· Accumulation of isocitrate and citrate occurs, but is limited by the inhibitory effects of high ATP and NADH.

· Citrate acts as an additional inhibitor to slow the fructose 6-phosphate reaction of glycolysis.

· The accumulated citrate also switches acetyl CoA to the synthesis of fatty acids.

Evolution has led to metabolic efficiency

· Glycolysis and fermentation are the most ancient energy-harvesting pathways.

· Some cells gained the capacity to perform photosynthesis, which has added oxygen to the atmosphere, rendering most environments aerobic.

· Cells evolved that could use oxygen, substantially improving their efficiency.

· Refinements occurred that improved the efficiency of the capture of energy from glucose to the remarkable level of 63 percent.

The Instructor’s Resource CD-ROM includes photographs of natural and genetically engineered bioluminescent organisms, an electron micrograph of a mitochondrion, and video microscopy of mitochondria in motion.