The Glycolytic Pathway

What are the primary components of a metabolic pathway?

● 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).

● Metabolism is hierarchical, consisting of 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 free energy delta G, which is calculated by taking into consideration the actual concentrations of substrates and products in the cell. Reactions with delta G < 0 are spontaneous in the forward direction, even if delta G°’ > 0 (nonspontaneous) 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 free energy changes is less than zero. Coupled reactions in cells often involve use of the phosphoryl transfer energy available in ATP (delta G°’ = -30.5 kJ/mol).

Why are monosaccharides and disaccharides called 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.

● 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).

● Disaccharides contain two simple sugars covalently linked by an O-glycosidic bond, which can be either an α or β glycosidic bond. Maltose contains two glucose molecules linked by an α(1–>4) glycosidic bond, and lactose contains glucose and galactose molecules linked by a β(1–>4) glycosidic bond.

What are the key components of the Glycolytic Pathway?

● 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, pyruvate kinase) catalyze highly favorable reactions (DG V 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 free 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.

What regulates metabolic flux through the Glycolytic Pathway?

● 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 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 sitelocated 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 signals a high energy charge in the cell, is an allosteric inhibitor of pyruvate kinase.

What is the metabolic fate of pyruvate?

● 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.

● 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.