Introduction and Goals
This tutorial describes the breakdown of glucose into carbon dioxide and water. In cells, this occurs in a stepwise fashion that starts in the cytoplasm and ends in the mitochondria. As chemical bonds are broken, energy is captured and stored in the form of high-energy molecules. There are three major stages in the breakdown of glucose. In the first stage, six-carbon sugars are cleaved into three-carbon sugars. In the second stage, these three-carbon sugars are further broken down in two alternate pathways: one that requires oxygen and one that does not. In the third and final stage, sugars are further broken down, subsequently releasing carbon dioxide and generating more high-energy molecules.
By the end of this tutorial you should know:
- The three phases and net yield of glycolysis
- The oxidation and reduction reactions, especially the role of electron carriers
- The allosteric regulation of glycolysis
- The role of pyruvate as a branching point for different pathways
- The pathways of lactate fermentation and ethanol fermentation
- The citric acid cycle, especially the first step, last step and products
- The role of the citric acid cycle in other metabolic pathways
Overview of Cellular Respiration
All cells require some source of energy to carry out their normal functions. The energy in cells is usually stored in the form of chemical bonds. In the next few tutorials you will learn about metabolic pathways (pathways of chemical reactions in a cell), including catabolic pathways, which describe reactions that breakdown molecules, and anabolic pathways, which describe reactions that build molecules. Often catabolic pathways release energy when chemical bonds are broken, whereas anabolic pathways may require energy to form chemical bonds. In plant cells, energy is derived from sunlight and used in anabolic pathways to synthesize simple sugars. These sugars can be stored and used later in either anabolic or catabolic pathways. In animal cells, energy is derived from the catabolism of ingested macromolecules such as starch and fat from other organisms (e.g. the hamburger you had for lunch). The large macromolecules are catabolized into simple sugars and other building blocks, releasing energy along the way. This energy is captured in the form of two types of high-energy molecules: ATP and electron carriers.
This tutorial describes the catabolism of glucose, the most common simple sugar found in both animals and plants. Remember from a previous tutorial (Properties of Macromolecules II: Nucleic Acids, Polysaccharides and Lipids), glucose is found in both glycogen and starch. The complete catabolism of glucose into CO2 and H2O is referred to as cellular respiration because it requires oxygen. The net reaction for cellular respiration is C6H12O6 + 6O2 -> 6CO2 + 6H2O + 38ATP. The catabolism of glucose occurs through a series of oxidation reactions. Recall from Biology 110 that the oxidationof a molecule involves the removal of electrons. The oxidation of organic molecules occurs by the removal of electrons and protons (H+). In biological reactions, an oxidation reaction is coupled to a reduction reaction (the addition of electrons and protons) such that one molecule is oxidized and the other is reduced. In the catabolism of glucose, sugars are oxidized in reactions that are coupled to the reduction of the most common electron carrier, nicotinamide adenine dinucleotide (NAD+), (Figure 1). For instance, in the following reaction: malate + NAD+ -> oxaloacetate + NADH + H+, malate is oxidized and NAD->is reduced. Cellular respiration occurs in a stepwise fashion, initially producing many molecules of reduced electron carriers (NADH and FADH2). These reduced electron carriers will eventually be oxidized in the mitochondria in a process that is linked to ATP synthesis. It is only in this final step that oxygen is actually used. The reduced electron carriers donate their electrons to an electron transport chain, and eventually, oxygen is reduced to yield water. This final step of cellular respiration yields the largest amount of energy, in the form of ATP.
There are four distinct stages of cellular respiration: glycolysis, the oxidation of glucose to the three-carbon sugar pyruvate; pyruvate oxidation, the oxidation of pyruvate to acetyl coenzyme A (acetyl CoA); the citric acid cycle(also referred to as the Kreb's cycle or TCA cycle), the complete oxidation of acetyl CoA; and finally, the oxidation of the reduced electron carriers linked to the synthesis of ATP. The first three stages (glycolysis, pyruvate oxidation and the citric acid cycle) will be described in this tutorial. In addition, we will consider the process of fermentation, which occurs in the absence of oxygen, whereby pyruvate is reduced and a variety of by-products are generated. The final step of cellular respiration, the oxidation of the electron carriers linked to ATP synthesis, will be covered in the next tutorial.
The Glycolytic Pathway
Glycolysis is a ten-step pathway that cleaves each glucose molecule (a 6-carbon sugar) into two molecules of pyruvate (a 3-carbon sugar) and that yields two molecules of ATP and two molecules of NADH. The pathway occurs in the cytoplasm, where each step is catalyzed by a different enzyme. Rather than memorize each step of glycolysis, we will categorize them into three distinct phases: Phase I: preparation of glucose; Phase II: cleavage of a 6-carbon sugar; and Phase III: oxidation and ATP generation (see animation below). The first phase of glycolysis requires the investment of ATP to prepare glucose for cleavage. This seems contrary to the previous statement that glycolysis results in the synthesis of two molecules of ATP per molecule of glucose. However, although two ATPs are used, an additional four ATPs are synthesized, resulting in a net yield of two ATPs per glucose molecule. The hydrolysis of ATP to ADP and Pi provides both the energy and the phosphate group required to phosphorylate the 6-carbon sugar; first, in reaction #1, by converting glucose to glucose-6-phosphate, and then, in reaction #3, by converting fructose-6-phosphate to fructose-1,6-biphosphate. The second phase of glycolysis involves the cleavage of one molecule of fructose-1,6-biphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (in animation below, reaction 4), the latter of which is readily converted to glyceraldehyde-3-phosphate, resulting in two molecules of glyceraldehyde-3-phosphate. The third phase of glycolysis involves the oxidation of the 3-carbon sugars and the generation of NADH and ATP. In two steps (reaction 7 and reaction 10), ATP is synthesized by substrate-level phosphorylation; the high-energy phosphate bond on the 3-carbon sugar is broken and the phosphate is transferred to ADP to synthesize ATP. In this fashion, two molecules of ATP are synthesized for each glyceraldehyde-3-phosphate, and since there are two glyceraldehyde-3-phosphates generated per glucose, four molecules of ATP are synthesized per molecule of glucose. In addition, two molecules of NADH are generated per molecule of glucose. The reduced electron carrier NADH will further contribute to the synthesis of ATP in the mitochondria (reviewed in the next tutorial).
Regulation of Glycolysis
Many of the steps of glycolysis are reversible, and, in fact, gluconeogenesis, which is the anabolic pathway that synthesizes glucose from pyruvate, is essentially glycolysis run in reverse (Figure 2). Most of the steps of gluconeogenesis are catalyzed by the same enzymes as glycolysis, with the exception of three important reactions that are strongly exergonic and that drive glycolysis in the forward direction. In glycolysis these three reactions are step #1 (glucose + ATP -> glucose-6-phosphate + ADP), which is catalyzed by the enzyme hexokinase, step #3 (fructose-6-phosphate + ATP -> fructose-1,6-biphosphate), which is catalyzed by the enzyme phosphofructokinase 1 (PFK1), and the last step (phosphoenolpyruvate + ADP -> pyruvate + ATP), which is catalyzed by the enzyme pyruvate kinase. In gluconeogenesis these three reactions occur in the reverse direction and are catalyzed by different enzymes.
The three steps listed above for glycolysis are regulated by allosteric regulation. Recall from the tutorial entitled Enzyme Kinetics and Catalysis, allosteric regulation of enzyme activity occurs due to a conformational change induced by the binding of both allosteric activators and inhibitors. Regulation of the rate of these three strongly exergonic reactions affects the overall rate of glycolysis. Their enzymes are regulated by the products of glycolysis (e.g. pyruvate kinase is activated by fructose-1,6-biphosphate), the products of other stages of cellular respiration (e.g. citrate, a product of the citric acid cycle, which inhibits phosphofructokinase 1) and the overall ratio of ADP/ATP (e.g. ATP inhibits phosphofructokinase 1, whereas ADP activates it). This mechanism balances the rate of glycolysis with the overall rate of cellular respiration and ATP synthesis. Therefore, when cellular respiration is running well and the levels of intermediates (e.g. citrate and acetyl CoA) and ATP are high, the rate of glycolysis is reduced. Conversely, when citrate, acetyl CoA and ATP levels are low, the rate of glycolysis is increased.
Another important allosteric activator of phosphofructokinase 1 is fructose-2-6-biphosphate (F2,6BP), which is generated from fructose-6-phosphate by the enzyme phosphofructokinase 2. Phosphofructokinase 2 (PFK-2) is a bifunctional enzyme that acts as a phosphatase or kinase, depending on its phosphorylation state, which is determined by hormone-regulated signal transduction cascades. In response to insulin production (activated by high blood sugar levels), PFK-2 is unphosphorylated and the kinase is activated, generating F2,6BP. F2,6BP activates phosphofructokinase 1 and stimulates glycolysis, thereby reducing the high levels of glucose. See Figure 2 for a summary of the allosteric regulation of glycolysis.
Pyruvate as a Branching Point
The end-point of glycolysis is the formation of pyruvate (2 molecules of pyruvate per molecule of glucose), which can enter several different metabolic pathways depending on the type of organism and the presence of oxygen. In the presence of oxygen, pyruvate enters the remaining stages of cellular respiration. Pyruvate is oxidized in a reaction that generates acetyl CoA, NADH and CO2 (Figure 3). Acetyl CoA is further oxidized to CO2 and H2O in the citric acid cycle (described in detail below).
In organisms that can grow in the absence of oxygen (anaerobic organisms) and in aerobic organisms (oxygen-using organisms) that can grow when oxygen is insufficient, pyruvate has an alternative fate. Under these conditions pyruvate undergoes a process termed fermentation, whereby pyruvate is reduced and NADH is oxidized to regenerate NAD+. The regeneration of NAD+ is critical for the ability of the cell to undergo additional rounds of glycolysis and to generate additional energy in the form of ATP. Depending on the cell type, there are two types of fermentation reactions: lactate fermentation and alcohol fermentation (illustrated in Figure 3). When there is insufficient oxygen in muscles, pyruvate is converted to lactate. In some organisms (e.g. yeast) that can grow anaerobically, pyruvate is converted to ethanol and CO2. We enjoy the by-products of alcohol fermentation in the bread we eat and the alcoholic beverages we drink. Note, although fermentation allows the cell to continue to undergo glycolysis, the net energy yield from fermentation is much lower than that from cellular respiration. Fermentation does not yield any additional energy, so under anaerobic conditions the yield of ATP is only two ATPs/glucose. The yield of ATP for complete cellular respiration is thirty-eight nucleotides per molecule of glucose (two ATPs from glycolysis and an additional thirty-six ATPs from subsequent reactions; which will be described later in this tutorial and the next tutorial).
The Citric Acid Cycle
The citric acid cycle, which takes place in the mitochondria, is the third stage of cellular respiration and it completes the oxidation of glucose. Recall that in glycolysis, glucose is converted to two molecules of pyruvate, and then pyruvate is further oxidized to acetyl CoA. In the citric acid cycle, acetyl CoA is completely oxidized to CO2 and reduced electron carriers are generated in the form of NADH and another molecule, flavin adenine dinucleotide (FAD). In addition, ATP is generated through substrate-level phosphorylation. The complete citric acid cycle is illustrated in Figure 4. In this tutorial we will focus on the first and last step, and the products of the citric acid cycle.
Acetyl CoA is the entry point to the citric acid cycle, and while acetyl CoA will be oxidized and CO2released, this does not happen directly but occurs via an eight-step process. The first step of the citric acid cycle is the transfer of two carbons from acetyl CoA to the 4-carbon sugar oxaloacetate to generate the 6-carbon sugar citrate- hence, the name of the cycle. This first step seems contrary to the purpose of the citric acid cycle (sugar oxidation); however, in the subsequent step of the cycle, sugars are oxidized and carbon bonds are cleaved to release two molecules of CO2, three molecules of NADH, one molecule of FADH2, and one molecule of ATP. The end product of the citric acid cycle is oxaloacetate, which you should recall combines with acetyl CoA to start the cycle. These reactions are referred to as a cycle because oxaloacetate is used in the first step and is regenerated in the last step. The citric acid cycle is analogous to a hand-cranked generator, where one turn of the crank produces energy in the form of electricity, while the crank itself is unaltered. That is, no new energy is generated, rather it is transformed from one form to another; therefore, the hand turning the crank provides the energy that will be converted to electricity. Using this analogy, the citric acid cycle is the generator, acetyl CoA provides the energy to turn the crank, and the energy of the carbon bonds are converted to the reduced electron carriers and ATP (analogous to the electricity).
The level of acetyl CoA is critical to driving the citric acid cycle. The first step (oxaloacetate + acetyl CoA -> citrate) is strongly exergonic. In addition, it keeps the oxaloacetate levels low, driving the last step of the cycle (malate + NAD+ -> oxaloacetate + NADH + H+). So far we have considered acetyl CoA derived from pyruvate oxidation, however, there are other sources of acetyl CoA. In fact, an important source is the oxidation of fatty acids (recall the tutorial Properties of Macromolecules II: Nucleic Acids, Polysaccharides and Lipids), which are the macromolecules that store the most energy.
Finally, the citric acid cycle is not solely linked to cellular respiration. It is, in fact, amphibolic (both anabolic and catabolic). Many of the intermediates of the cycle are siphoned off and used in other pathways. For instance, citrate is used in pathways to synthesize fatty acids and cholesterol. Several intermediates, including oxaloacetate, are precursors of amino acids. In order to be able to run the citric acid cycle efficiently, there are pathways that replenish the intermediates of the cycle as well.
Cellular respiration is a catabolic pathway in which glucose is completely oxidized, yielding CO2 and the high-energy, reduced electron carriers NADH and FADH2, and ATP. This tutorial reviewed the first three stages of cellular respiration: glycolysis, pyruvate oxidation and the citric acid cycle. Figure 5illustrates the net yield of ATP and reduced electron carriers for each of these stages. Glycolysis is the break down of glucose to give a net yield of two molecules of pyruvate, two ATPS and two NADH + H+. Glycolysis occurs in three phases: phase I: preparation of the sugar, which requires two ATPs to phosphorylate the 6-carbon sugar; phase II: cleavage of the 6-carbon sugar to two 3-carbon sugars; and phase III: oxidation of the sugars and generation of four ATPs and two NADH + H+ per glucose. Glycolysis is regulated by allosteric regulation of the enzymes hexokinase, phosphofructokinase and pyruvate kinase, which catalyze reactions at three steps that are highly exergonic. Gluconeogeneis is the generation of glucose, starting from pyruvate, and it is essentially glycolysis in reverse (with the important exception of three exergonic and highly regulated steps). Pyruvate is the end point of glycolysis and it is a branching point. In the absence of oxygen, pyruvate undergoes fermentation (either ethanol or lactate, depending on the organism). Fermentation does not generate any additional energy, however, NAD+ is regenerated. In the presence of oxygen, pyruvate is oxidized and acetyl CoA is formed, which feeds into the citrate acid cycle and the complete oxidation of glucose. The citric acid cycle begins when acetyl CoA combines with oxaloacetate to generate citric acid. One round of the cycle generates two CO2, three NAD+ + H+, FADH2and ATP, and oxaloacetate is regenerated. In addition to pyruvate, fatty acids are an important source of acetyl CoA. Finally, the citric acid cycle is amphibolic and is central to many other metabolic pathways.