Glycolysis Study Pack

Location and Universality

• •Glycolysis takes place entirely in the cytosol — the liquid interior of the cell — and requires no membrane-bound organelles.
• •Because it does not depend on mitochondria or oxygen, glycolysis is the only ATP-producing pathway available to cells under anaerobic conditions and to cells that lack mitochondria.
• •Its presence in bacteria, archaea, and eukaryotes alike reflects its evolutionary age and fundamental importance.

Starting Material and Overall Reaction

• •The sole carbon input is one molecule of glucose (C₆H₁₂O₆), a six-carbon sugar.
• •The pathway breaks that molecule into two molecules of pyruvate (C₃H₄O₃), each containing three carbons.
• •The overall transformation can be summarized as: glucose + 2 NAD⁺ + 2 ADP + 2 Pᵢ → 2 pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O.

Role Within Broader Cellular Respiration

• •Glycolysis is the first stage of cellular respiration but is not the same as cellular respiration; it feeds pyruvate and NADH into subsequent pathways.
• •Under aerobic conditions, pyruvate moves into the mitochondrial matrix for pyruvate oxidation and the citric acid cycle.
• •Under anaerobic conditions, pyruvate instead enters fermentation pathways that regenerate NAD⁺, allowing glycolysis to continue.

• •Step 1 — Hexokinase Phosphorylates Glucose
• •Hexokinase transfers a phosphate group from ATP to carbon-6 of glucose, producing glucose-6-phosphate and consuming the first ATP.
• •The added phosphate traps glucose inside the cell because the charged molecule cannot cross the plasma membrane, and it prevents the molecule from being shunted into other pathways.
• •Step 2 — Phosphoglucose Isomerase Converts to Fructose-6-Phosphate
• •Phosphoglucose isomerase rearranges glucose-6-phosphate into fructose-6-phosphate, an isomeric six-carbon sugar phosphate.
• •This conversion is necessary because the subsequent phosphorylation and cleavage reactions are specific to the fructose configuration.
• •Step 3 — Phosphofructokinase-1 Commits the Molecule
• •Phosphofructokinase-1 (PFK-1) adds a second phosphate group from ATP to carbon-1 of fructose-6-phosphate, generating fructose-1,6-bisphosphate and consuming the second ATP.
• •This step is essentially irreversible under cellular conditions and is the most tightly regulated step in glycolysis, making it the committed step of the pathway.
• •Steps 4 and 5 — Cleavage and Isomerization Yield Two Three-Carbon Units
• •Aldolase cleaves fructose-1,6-bisphosphate into two different three-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).
• •Triose phosphate isomerase rapidly and reversibly converts DHAP into G3P, so two molecules of G3P continue through the remaining steps.
• •At the end of the investment phase, the cell has spent 2 ATP and holds two molecules of G3P ready for energy extraction.

• •Step 6 — Glyceraldehyde-3-Phosphate Dehydrogenase Oxidizes G3P
• •Glyceraldehyde-3-phosphate dehydrogenase oxidizes each G3P molecule and simultaneously adds an inorganic phosphate (Pᵢ) from the cytosol, producing 1,3-bisphosphoglycerate (1,3-BPG).
• •During this oxidation, each NAD⁺ accepts two electrons and a proton to become NADH — yielding 2 NADH total across both G3P molecules.
• •The energy released by oxidation is captured in the high-energy phosphate bond of 1,3-BPG rather than lost as heat.
• •Step 7 — Substrate-Level Phosphorylation Produces ATP (First Time)
• •Phosphoglycerate kinase transfers the high-energy phosphate from 1,3-BPG directly to ADP, producing 3-phosphoglycerate and generating 1 ATP per molecule.
• •Because two molecules of 1,3-BPG are processed, this step yields 2 ATP total, which exactly cancels the 2 ATP spent during the investment phase.
• •Steps 8 and 9 — Rearrangement Prepares for Final ATP Synthesis
• •Phosphoglycerate mutase moves the remaining phosphate group from carbon 3 to carbon 2, producing 2-phosphoglycerate.
• •Enolase removes a water molecule from 2-phosphoglycerate, forming phosphoenolpyruvate (PEP), a molecule with an especially high-energy phosphate bond.
• •Step 10 — Pyruvate Kinase Completes Glycolysis
• •Pyruvate kinase transfers the phosphate from PEP to ADP, generating pyruvate and the final 2 ATP (1 per molecule, 2 total).
• •This step is irreversible under cellular conditions and, like PFK-1, is a key regulatory point.
• •The net result after both phases is 2 pyruvate, 2 ATP (net), and 2 NADH per original glucose molecule.