Glycolysis: Steps and Regulation

• Glycolysis is a metabolic pathway that converts glucose into pyruvate, producing ATP and NADH.
• Irreversible steps in glycolysis (1, 3, and 10) are major regulatory points.
• Overall reaction: Glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 Pyruvate + 2 ATP + 2 NADH + 2 H2O

Steps in Glycolysis

• Step 1: Glucose → Glucose-6-phosphate, enzyme: Hexokinase/Glucokinase (Transferase), irreversible.
• Step 2: Glucose-6-phosphate ⇌ Fructose-6-phosphate, enzyme: Phosphoglucose isomerase (Isomerase), reversible.
• Step 3: Fructose-6-phosphate → Fructose-1,6-bisphosphate, enzyme: Phosphofructokinase-1 (Transferase), irreversible.
• Step 4: Fructose-1,6-bisphosphate ⇌ Dihydroxyacetone phosphate (DHAP) + Glyceraldehyde-3-phosphate (G3P), enzyme: Aldolase (Lyase), reversible.
• Step 5: DHAP ⇌ G3P, enzyme: Triose phosphate isomerase (Isomerase), reversible.
• Step 6: G3P + Pi + NAD+ ⇌ 1,3-bisphosphoglycerate + NADH, enzyme: G3P dehydrogenase (Oxidoreductase), reversible.
• Step 7: 1,3-bisphosphoglycerate + ADP ⇌ 3-phosphoglycerate + ATP, enzyme: Phosphoglycerate kinase (Transferase), reversible.
• Step 8: 3-phosphoglycerate ⇌ 2-phosphoglycerate, enzyme: Phosphoglycerate mutase (Isomerase), reversible.
• Step 9: 2-phosphoglycerate ⇌ Phosphoenolpyruvate (PEP), enzyme: Enolase (Lyase), reversible.
• Step 10: PEP + ADP → Pyruvate + ATP, enzyme: Pyruvate kinase (Transferase), irreversible.
• Converting glucose to glucose-6-phosphate prevents glucose from diffusing out of the cell and commits it to further metabolism (glycolysis or glycogenesis).

Irreversible Steps

• Steps 1, 3, and 10 are irreversible and act as regulatory checkpoints.
• Hexokinase, Phosphofructokinase-1 (PFK-1), and Pyruvate Kinase control glycolysis flux.

Hexokinase vs Glucokinase

• Km (Affinity for Glucose): Hexokinase has low Km (high affinity), while Glucokinase has high Km (low affinity).
• Vmax: Hexokinase has low Vmax, while Glucokinase has high Vmax.
• Location: Hexokinase is in most tissues, while Glucokinase is in the liver and pancreas.
• Hexokinase allows tissues to utilize glucose even at low concentrations.
• Glucokinase helps the liver store glucose when levels are high.

Committed Step

• A committed step is an irreversible enzymatic reaction that locks the substrate into a specific metabolic pathway, which occurs at step 3.
• Phosphofructokinase-1 (PFK-1) catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate.
• After step 5, 2 molecules of G3P are produced per molecule of glucose

ATP Production

• Step 7: 1,3-bisphosphoglycerate (1,3-BPG) → 3-phosphoglycerate (ATP generated).
• Step 10: Phosphoenolpyruvate (PEP) → Pyruvate (ATP generated).
• Lactate is produced to regenerate NAD+ allowing glycolysis to continue in the absence of oxygen, essential for ATP generation in anaerobic conditions (e.g., intense exercise in muscles).

Lactate

• Lactate can be transported to the liver for gluconeogenesis via the Cori Cycle.
• Lactate can be used as an energy source by the heart and other tissues.
• Glucose to pyruvate (Aerobic Glycolysis) yields a net of 2 ATP per glucose.
• Glucose to lactate (Anaerobic Glycolysis) yields a net of 2 ATP per glucose, with NADH recycled.
• Aerobic conditions: NADH is shuttled to the mitochondria for ATP production via the malate-aspartate shuttle or glycerol-3-phosphate shuttle.
• Anaerobic conditions: NADH is used to reduce pyruvate to lactate, regenerating NAD+ for glycolysis.

Enzymes

• Alanine transaminase (ALT) catalyzes the reaction: Pyruvate + Glutamate → Alanine + α-Ketoglutarate
• Pyruvate is converted to ethanol in yeast to regenerate NAD+ for glycolysis, and to allow ATP production under anaerobic conditions.

Pyruvate Dehydrogenase

• Pyruvate dehydrogenase is located in the mitochondrial matrix.
• Thiamine pyrophosphate (TPP) - Decarboxylation of pyruvate.
• Lipoic acid - Transfers acetyl group.
• FAD (Flavin Adenine Dinucleotide) - Regenerates NADH.
• Each NADH generated by pyruvate dehydrogenase enters the electron transport chain, yielding ~2.5 ATPs.

ATP Production

• Pyruvate → Acetyl-CoA: 5 ATP (2 NADH = 5 ATP)
• TCA Cycle: 20 ATP (2 Acetyl-CoA → 6 NADH, 2 FADH2, 2 GTP)
• Total: 25 ATP per glucose (from pyruvate stage onward)
• The complete oxidation from glycolysis yeilds 7 ATP

Multi-Enzyme Complex

• Multi-enzyme complex consists of multiple enzymes that work together to catalyze sequential reactions, enhancing metabolic efficiency by preventing loss of intermediates and increasing reaction speed.
• It catalyzes the conversion of pyruvate into acetyl-CoA, linking glycolysis to the citric acid cycle.
• Pyruvate → Acetyl-CoA: 5 ATP
• Citric Acid Cycle: 20 ATP
• About 32 ATP per glucose generated

Citric Acid Cycle

• Citric acid cycle serves both catabolic (energy production) and anabolic (biosynthesis) functions, making it an amphibolic pathway.
• Anaplerotic reactions replenish TCA intermediates; Pyruvate carboxylase converts pyruvate into oxaloacetate.
• FADH2 donates electrons to the electron transport chain, generating 1.5 ATP per molecule.
• It is regulated by substrate availability, allosteric enzymes (e.g., citrate synthase, isocitrate dehydrogenase), and energy status (ATP/NADH levels).
• A variation of the TCA cycle found in plants and bacteria that enables the conversion of acetyl-CoA to glucose, bypassing CO2-producing steps is the glyoxylate cycle.
• The pentose phosphate pathway produces NADPH (for biosynthesis) and ribose-5-phosphate (for nucleotide synthesis).
• Gluconeogenesis reverses glycolysis, but with different enzymes for irreversible steps (e.g., pyruvate carboxylase instead of pyruvate kinase).
• Gluconeogenesis is activated by glucagon and inhibited by insulin.

Cori Cycle

• Transports lactate from muscles to the liver, where it is converted back to glucose to prevent lactic acidosis and sustain ATP production.

Key Regulatory Enzymes

• The key regulatory enzymes in glycolysis: Hexokinase, Phosphofructokinase-1 (PFK-1), Pyruvate Kinase.
• Gluconeogenesis: Pyruvate Carboxylase, PEP Carboxykinase, Fructose-1,6-bisphosphatase, Glucose-6-phosphatase.
• High ATP inhibits glycolysis by inhibiting PFK-1 and pyruvate kinase.
• Low ATP (high AMP) activates glycolysis and inhibits gluconeogenesis.
• Glycogenesis: The synthesis of glycogen from glucose.
• Glycogenolysis: The breakdown of glycogen into glucose.
• Glycogenesis: Glycogen Synthase, Branching Enzyme.
• Glycogenolysis: Glycogen Phosphorylase, Debranching Enzyme.

Glycogen Metabolism

• Insulin activates glycogenesis (storage of glucose as glycogen).
• Glucagon and epinephrine activate glycogenolysis (breakdown of glycogen to release glucose).
• UDP-glucose is the activated form of glucose that serves as a direct substrate for glycogen synthase.
• Insulin activates glycogen synthase and inhibits glycogen phosphorylase, leading to glucose storage as glycogen.
• Glucagon stimulates gluconeogenesis and glycogenolysis and inhibits glycolysis and glycogenesis.
• Skeletal muscle cells lack glucagon receptors and rely on epinephrine for glycogen breakdown.
• Liver cells respond to glucagon to regulate blood glucose levels.

Carbohydrate Metabolism

• Activates PFK-1 (glycolysis) and inhibits Fructose-1,6-bisphosphatase (gluconeogenesis).
• Liver: Glucokinase (high Km, functions when glucose is high).
• Muscle: Hexokinase (low Km, active at all glucose levels).
• Phosphorylation of Pyruvate Kinase (by glucagon): Inactivates glycolysis.
• Dephosphorylation (by insulin): Activates glycolysis.
• The key enzyme glucose-6-phosphate dehydrogenase (G6PD) is inhibited by high levels of NADPH and activated by NADP+.
• Oxidative phase: Generates NADPH.
• Non-oxidative phase: Produces ribose-5-phosphate and glycolysis intermediates.
• NADPH from the pentose phosphate pathway is required for fatty acid synthesis.
• Acetyl-CoA from glucose metabolism is used for lipogenesis.
• AMPK is activated by low ATP levels.
• It stimulates glucose uptake, fatty acid oxidation, and inhibits gluconeogenesis.
• Excess glucose is stored as glycogen (glycogenesis) or converted to fatty acids (lipogenesis).
• Cancer cells favor aerobic glycolysis (high glucose uptake, lactate production) even in the presence of oxygen to support rapid proliferation (Warburg Effect).

Diabetes

• Type 1 Diabetes: Lack of insulin leads to increased gluconeogenesis, glycogenolysis, and hyperglycemia.
• Type 2 Diabetes: Insulin resistance leads to impaired glucose uptake and chronic hyperglycemia.
• Summary of Regulation of Carbohydrate Metabolism*

###Liver Metabolism

• High blood glucose level, high [insulin], low glucagon: Glycolysis, Pentose Phosphate Pathway, Glycogenesis are active.

• Glucose-6-Phosphate is converted into pyruvate (glycolysis), used for NADPH and ribose synthesis (PPP), or stored as glycogen.

• Low blood glucose level, low [insulin], high [glucagon]: Gluconeogenesis, Glycogenolysis are active.

• Glucose-6-Phosphate is used to generate free glucose for release into the bloodstream. ###Muscle Metabolism

• High blood glucose level, high [insulin]: Glycogenesis is active. Glucose-6-Phosphate is stored as glycogen for later use.

• High blood glucose level, high [insulin]: Glycolysis, Glycogenolysis are active. Glucose-6-Phosphate is used for ATP production via glycolysis or broken down from glycogen for energy.