The Glycolytic Pathway

How do cells direct the flow of metabolites through coupled catabolic and anabolic pathways?

• Catabolic pathways degrade macromolecules and nutrients to capture energy in the form of ATP and reduction potential. Anabolic pathways use this energy to synthesize biomolecules for the cell.

• Metabolic flux refers to the rate at which metabolites are interconverted in catabolic and anabolic pathways. It is determined by (1) availability of substrates and (2) amount of enzyme activity (enzyme level and catalytic activity).

• Metabolic pathways are interdependent and tightly regulated, and flux through them is continuous, not all-or-none. Even opposing catabolic and anabolic pathways operate simultaneously. What changes is the relative rate in each pathway as regulatory signals shift metabolite flow in one direction or the other.

• Metabolism requires macromolecules (proteins, nucleic acids, carbohydrates, and lipids), primary metabolites (amino acids, nucleotides, fatty acids, glucose, pyruvate, and acetyl-CoA), and small molecules (NH₄⁺, CO₂, NADH, FADH₂, O₂, ATP, and H₂O).

• The spontaneity of metabolic reactions is determined by the change in actual Gibbs energy ΔG, which is calculated by taking into consideration the actual concentrations of substrates and products in the cell. Reactions with ΔG < 0 are spontaneous (thermodynamically favorable) in the forward direction, even if ΔG°′ > 0 (nonspontaneous—that is, thermodynamically unfavorable) for the same reaction.

• Coupled reactions in a pathway provide a mechanism to overcome unfavorable individual reactions if the total sum of all individual biochemical standard Gibbs energy changes is less than zero. Coupled reactions in cells often involve use of the phosphoryl transfer energy available in ATP (ΔG°′ = −30.5 kJ/mol).

• Protein complexes containing multiple enzymes for a related set of pathway reactions provide one way to facilitate coupled reactions, because they decrease loss of shared intermediates due to diffusion. Some protein complexes are built around scaffold proteins that function as organizing centers.

What are the structures and chemical properties of simple sugars?

• Simple sugars are metabolites that feed into the glycolytic pathway. They include aldose sugars, such as glucose, and ketose sugars, such as fructose.

• Glucose has the molecular formula C₆H₁₂O₆ and is a polyhydroxyaldehyde, whereas fructose, a monosaccharide with the same molecular formula as glucose, is a polyhydroxyketone.

• Aldose sugars in the open-chain conformation can be oxidized to carboxylic acids in a redox reaction with copper (Cu²⁺ → Cu⁺), a reaction known as Benedict’s test. Sugars that react with Cu²⁺ are called reducing sugars (for example, glucose, galactose, and lactose), whereas unreactive sugars are nonreducing (for example, sucrose, trehalose, and raffinose).

• The smallest monosaccharide is glyceraldehyde, which is a C₃ sugar with one chiral center (a carbon bonded to four different chemical groups). Most monosaccharides such as glucose and fructose have the D conformation, although L conformations do exist in nature.

• Cyclic monosaccharides form spontaneously through a covalent linkage of the carbonyl carbon with a hydroxyl group in the carbon backbone. If this bond is between alcohol and aldehyde groups, then it forms a hemiacetal (aldose sugar). If it is between alcohol and ketone groups, then it forms a hemiketal (ketose sugar).

• When the cyclic form of glucose has the hydroxyl group at C-1 on the same side of the ring as the CH₂OH at C-6, it is β-D-glucose, whereas when the hydroxyl group at C-1 is on the opposite side of the ring as the CH₂OH, it is α-D-glucose.

• Modern glucose sensors use immobilized glucose oxidase enzyme on the surface of a subcutaneous electrode that converts glucose in the interstitial fluid to H₂O₂ and, in the process, generates an electric current (H₂O₂ → O₂ + 2 H⁺ + 2 e⁻) that can be measured and converted to glucose concentration.

• Disaccharides contain two simple sugars covalently linked by an O-glycosidic bond, which consists of α and/or β cyclic sugar conformations. The same rules apply for naming the α and β conformations of glycosidic bonds as in cyclic sugars, which relies on the position of the O(H) group at the C-1 carbon being on the same side of the ring (β conformation) or opposite side of the ring (α conformation) relative to the CH₂OH group at C-6.

• The hemiacetal C-1 of cyclic D-glucose is an anomeric carbon; β-D-glucose and α-D-glucose are referred to as anomers because they differ only at the anomeric carbon. Cyclic conformations of sugars are pyranoses because the ring is similar to pyran, a cyclic C₅H₆O compound.

How does glycolysis break down glucose to generate ATP under anaerobic conditions?

• Glycolysis is a core metabolic pathway for three reasons: (1) glycolytic enzymes are highly conserved; (2) glycolysis is the primary pathway for ATP generation under anaerobic conditions; and (3) glycolytic intermediates are shared metabolites in a variety of interconnected pathways.

• Glycolysis converts one molecule of glucose to two molecules of pyruvate with no loss of carbon or oxygen atoms. It generates two net ATP molecules and two NADH molecules for every glucose molecule that is metabolized.

• The glycolytic pathway consists of 10 enzymatic reactions: five reactions in stage 1, the ATP investment phase, and five reactions in stage 2, the ATP earnings phase. Because two molecules of glyceraldehyde-3-P are generated for every molecule of glucose that is metabolized, all of the reactions in stage 2 occur twice for each glucose molecule that enters the pathway.

• Three glycolytic enzymes (hexokinase, phosphofructokinase-1, and pyruvate kinase) catalyze highly favorable reactions (ΔG ≪ 0) in glycolysis and are regulated to control flux through the pathway. The other seven enzymes catalyze readily reversible reactions that are shared with the gluconeogenic pathway.

• Substrate-level phosphorylation reactions generate ATP by direct transfer of a phosphoryl group from a donor to ADP. Phosphoryl donors in substrate-level phosphorylation reactions have biochemical standard Gibbs energy changes of phosphate hydrolysis that are more negative than that of ATP hydrolysis.

• Reaction 10 in the glycolytic pathway is catalyzed by the enzyme pyruvate kinase, which uses substrate-level phosphorylation to transfer the phosphoryl group from phosphoenolpyruvate to ADP. Because this reaction occurs twice for every glucose molecule that enters the glycolytic pathway, a net of 2 ATP is generated.

How is flux through the glycolytic pathway regulated to meet cellular energy needs and control blood glucose levels?

• Glucokinase is an isozyme of hexokinase that converts glucose to glucose-6-P in pancreatic and liver cells. Unlike hexokinase, however, glucokinase has a very low affinity for glucose (100 times lower) and is not feedback inhibited by glucose-6-P, which results in its activity being controlled by physiologic glucose levels.

• Liver glucokinase is regulated by cellular localization through the binding of glucokinase regulatory protein (GKRP), which binds to glucokinase under conditions of low intracellular glucose levels and sequesters glucokinase in the nucleus. Elevated glucose levels in liver cells outcompete GKRP for binding to glucokinase in the nucleus and free up glucokinase to export to the cytoplasm and stimulate glycolytic flux.

• Increased blood glucose levels stimulate glucokinase activity in pancreatic β cells. This leads to increased flux through the glycolytic pathway, elevated ATP levels, and ultimately Ca²⁺-mediated stimulation of insulin release from the β cells. This glucose-sensing function of glucokinase is crucial to maintaining safe homeostatic levels of glucose in the blood.

• Phosphofructokinase-1 (PFK-1) is allosterically regulated by metabolites in the cell that signal changes in the energy charge and flux through the glycolytic and citrate cycle pathways. PFK-1 is activated by AMP, ADP, and fructose-2,6-bisphosphate and is inhibited by ATP and citrate.

• ADP, AMP, and ATP bind to an allosteric effector site located at the interface of two PFK-1 subunits. ADP and AMP bind with high affinity to the active R-state conformation and stabilize it, whereas ATP binds to the same site when the protein complex is in the inactive T-state conformation, leading to inhibition of PFK-1 activity.

• Pyruvate kinase is allosterically activated by fructose-1,6-bisphosphate, which binds to the enzyme and stabilizes the active tetrameric form to mediate feed-forward regulation. Increased levels of ATP, which signal a high energy charge in the cell, are an allosteric inhibitor of pyruvate kinase.

• ATP serves as both a substrate at the catalytic site and an allosteric regulator of PFK-1 at a distinct regulatory site. Binding of ATP at the catalytic site is mechanistically separate from ATP or ADP binding to the regulatory allosteric site.

• In addition to glucose, fructose and galactose can also be converted to glycolytic intermediates and used to generate ATP. The disaccharide sucrose contains a glucose molecule linked to fructose and is hydrolyzed by the enzyme sucrase, whereas lactose contains a glucose molecule linked to galactose and is hydrolyzed by the enzyme lactase.

• Lactose intolerance is a natural condition in adult humans in which the level of lactase in the intestine decreases with age, resulting in gastrointestinal problems when large quantities of dairy products containing lactose are ingested. Intestinal anaerobic bacteria ferment the undigested lactose to produce H₂ and CH₄ gases.

• Under high energy charge conditions, glycolytic intermediates provide metabolites for anabolic pathways. For example, glucose-6-P is converted to pentose phosphates and used for nucleotide biosynthesis or the production of NADPH by the pentose phosphate pathway. Several C₃ metabolites derived from stage 2 glycolytic reactions provide precursor molecules for amino acid biosynthesis.

What are the metabolic fates of pyruvate under aerobic and anaerobic conditions?

• Pyruvate is metabolized under aerobic conditions in the mitochondria to acetyl-CoA and ultimately to CO₂ and H₂O by the citrate cycle and electron transport chain, generating the bulk of ATP derived from glucose metabolism.

• Under anaerobic conditions, such as in exercising muscle or in microorganisms when O₂ levels in the environment are low, pyruvate is reduced by the enzyme lactate dehydrogenase to produce lactate. The yeast Saccharomyces cerevisiae uses alcoholic fermentation under anaerobic conditions to convert pyruvate to CO₂ and ethanol.

• Mutations in the mitochondrial aldehyde dehydrogenase gene (ALDH2) can lead to severe reactions owing to excessive alcohol consumption. In the short term, these symptoms include facial flushing, tachycardia (increased heart rate), headache, and nausea, whereas chronic alcohol abuse leads to an increased risk of esophageal cancer and cardiovascular disease.

• Defects in the enzyme lactate dehydrogenase cause the genetic disease LDH deficiency, which is characterized by an inability to sustain intense muscle activity under anaerobic conditions. LDH deficiency results in decreased flux through the glyceraldehyde-3-phosphate dehydrogenase reaction when NAD⁺ levels are depleted and ATP production by the glycolytic pathway is limited.

• A critical function of pyruvate metabolism is to replenish NAD⁺ levels in the cytoplasm in order to maintain flux through the glyceraldehyde-3-P dehydrogenase reaction. Under anaerobic conditions, this is done by the enzymes lactate dehydrogenase and alcohol dehydrogenase, whereas under aerobic conditions, mitochondrial shuttle systems in eukaryotic cells are required.