Glycolysis: Overview and Stages

Overview of Glycolysis

• This metabolic pathway converts glucose to pyruvate, generating ATP and NADH
• It takes place in the cell's cytoplasm
• Glycolysis enzymes are organized sequentially for efficient substrate transfer

Two Stages of Glycolysis

• Stage 1 is the Energy Investment Phase
• Glucose is phosphorylated and split into two 3-carbon molecules
• This stage requires ATP input
• Stage 2 is the Energy Payoff Phase
• ATP and NADH are produced through substrate-level phosphorylation
• Each reaction happens twice per glucose molecule

Steps in Stage 1 of Glycolysis

• Hexokinase catalyzes the phosphorylation of glucose to glucose-6-phosphate, using ATP
• Phosphoglucose isomerase converts glucose-6-phosphate into fructose-6-phosphate
• Phosphofructokinase-1 (PFK-1) phosphorylates fructose-6-phosphate to fructose-1,6-bisphosphate, using ATP
• Aldolase splits fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)
• Triose phosphate isomerase converts DHAP into G3P
• One 6-carbon glucose is converted into two 3-carbon molecules (G3P)

Steps in Stage 2 of Glycolysis

• Glyceraldehyde 3-phosphate dehydrogenase oxidizes G3P to 1,3-bisphosphoglycerate, generating NADH
• Phosphoglycerate kinase converts 1,3-bisphosphoglycerate to 3-phosphoglycerate, producing ATP via substrate-level phosphorylation
• Phosphoglycerate mutase converts 3-phosphoglycerate into 2-phosphoglycerate
• Enolase converts 2-phosphoglycerate to phosphoenolpyruvate (PEP)
• Pyruvate kinase converts PEP to pyruvate, producing ATP via substrate-level phosphorylation
• Each step occurs twice per glucose molecule, leading to net ATP and NADH production

Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate

• Oxidation of G3P generates energy
• The energy attaches an inorganic phosphate, forming a high-energy acyl-phosphate intermediate
• This coupling thermodynamically favors the reaction

Enzymes and Their Functions

• Hexokinase phosphorylates glucose using ATP
• Phosphofructokinase-1 (PFK-1) is a major regulatory enzyme, adding a second phosphate to fructose-6-phosphate
• Aldolase splits fructose-1,6-bisphosphate into two 3-carbon sugars
• Glyceraldehyde 3-phosphate dehydrogenase produces NADH and forms acyl-phosphate
• Phosphoglycerate kinase generates ATP via substrate-level phosphorylation
• Pyruvate kinase converts PEP to pyruvate, producing ATP

Types of Reactions by Enzymes

• Kinases transfer phosphate groups (e.g., hexokinase, PFK-1, pyruvate kinase)
• Phosphatases remove phosphate groups
• Dehydrogenases catalyze oxidation-reduction reactions (e.g., glyceraldehyde 3-phosphate dehydrogenase)

Recognizing Phosphorylated Molecules

• The number of phosphates present can be recognized from the name (e.g., fructose-1,6-bisphosphate has 2 phosphates)

Substrate-Level Phosphorylation

• A direct transfer of a phosphate group to ADP forms ATP
• This occurs in the phosphoglycerate kinase reaction (1,3-BPG to 3-PG) and the pyruvate kinase reaction (PEP to pyruvate)

Net Reaction of Glycolysis

• Glucose + 2 NAD⁺ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 ATP + 2 H₂O + 2 H⁺

NAD⁺ Regeneration

• Essential for glycolysis to continue
• There are 3 pathways for pyruvate metabolism:
• Aerobic: Pyruvate enters the TCA cycle, and NADH donates electrons to the electron transport chain
• Lactic Acid Fermentation: Pyruvate becomes Lactate (via lactate dehydrogenase, regenerates NAD⁺)
• Ethanol Fermentation: Pyruvate becomes Acetaldehyde, which becomes Ethanol (regenerates NAD⁺, occurs in yeast)

Ethanol Fermentation

• Steps: Pyruvate → Acetaldehyde (via pyruvate decarboxylase, releases CO₂), then Acetaldehyde → Ethanol (via alcohol dehydrogenase, regenerates NAD⁺)
• Net Reaction: Glucose → 2 Ethanol + 2 CO₂ + 2 ATP
• Occurs in yeast and some bacteria

Lactic Acid Fermentation

• Steps: Pyruvate → Lactate (via lactate dehydrogenase, regenerates NAD⁺)
• Net Reaction: Glucose → 2 Lactate + 2 ATP
• Occurs in muscle cells during anaerobic respiration

Fructose Entry into Glycolysis

• Two pathways exist:
• In the liver: Fructose → Fructose-1-phosphate → DHAP + G3P (via fructokinase and aldolase B)
• In muscle: Fructose is directly phosphorylated to fructose-6-phosphate (via hexokinase)

Galactose Conversion to Glucose 6-Phosphate

• Steps: Galactose → Galactose-1-phosphate (via galactokinase), then Galactose-1-phosphate → UDP-galactose, then UDP-galactose → UDP-glucose, and finally UDP-glucose → Glucose-6-phosphate

Two Roles of Glycolysis

• This is the mechanism for energy production (ATP generation)
• Provides intermediates for biosynthetic pathways

Regulation of Glycolysis

• Three key enzymes regulate glycolysis:
  • Hexokinase is inhibited by glucose-6-phosphate
  • Phosphofructokinase-1 (PFK-1) is activated by AMP & fructose-2,6-bisphosphate and inhibited by ATP & citrate
  • Pyruvate kinase is activated by fructose-1,6-bisphosphate and inhibited by ATP & alanine
• Muscle regulation is based on energy demand
• Liver regulation maintains blood glucose levels

Glucose Transporters (GLUTs)

• GLUT1 is found in most cells for basal glucose uptake
• GLUT2 is in the liver and pancreas for bidirectional transport
• GLUT3 is in neurons as a high-affinity transporter
• GLUT4 is in muscle and fat for insulin-dependent uptake
• GLUT5 is a fructose transporter in the small intestine

Overview of Gluconeogenesis

• A metabolic pathway generating glucose from non-carbohydrate precursors
• Maintains blood glucose during fasting or prolonged exercise
• Occurs mainly in the liver, kidneys to a lesser extent

Precursors for Gluconeogenesis

• Lactate comes from anaerobic glycolysis in muscle, entering via the Cori cycle
• Amino acids (except leucine & lysine) are converted into intermediates of the TCA cycle
• Glycerol comes from triglyceride breakdown, entering as dihydroxyacetone phosphate (DHAP)

Irreversible Steps of Glycolysis That Are Bypassed

• Steps are bypassed involving:
  • Glucokinase/Hexokinase to Glucose-6-phosphatase
  • Phosphofructokinase-1 (PFK-1) to Fructose-1,6-bisphosphatase
  • Pyruvate kinase to Pyruvate carboxylase & PEP carboxykinase

First Step of Gluconeogenesis

• Pyruvate turns to Oxaloacetate (OAA)
• Pyruvate carboxylase catalyzes this, needing ATP and biotin
• This occurs in the mitochondria

Oxaloacetate Conversion and Transport

• OAA reduces to malate to cross the mitochondrial membrane, oxidizing back to OAA in the cytoplasm
• OAA becomes phosphoenolpyruvate (PEP) with PEP carboxykinase (PEPCK)
• The PEP conversion consumes GTP

Net Reaction of Pyruvate to PEP

• 2 Pyruvate + 2 ATP + 2 GTP → 2 PEP + 2 ADP + 2 GDP + 2 CO₂

Conversion of Fructose-1,6-bisphosphate to Fructose-6-phosphate

• Catalyzed by fructose-1,6-bisphosphatase

Conversion of Glucose-6-Phosphate to Free Glucose

• Glucose-6-phosphatase is the enzyme
• This occurs in the endoplasmic reticulum of liver and kidney cells
• Glucose-6-phosphate is transported into the ER for dephosphorylation, then free glucose is exported

Net Reaction of Gluconeogenesis

• 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH → Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD⁺
• Gluconeogenesis needs 6 ATP equivalents, making it more energy-intensive than glycolysis

Regulation of Glycolysis vs. Gluconeogenesis

• Mutual regulation is present, which prevents the pathways from operating simultaneously
• Key regulators include:
  • High ATP & citrate stimulate gluconeogenesis and inhibit glycolysis
  • High AMP stimulates glycolysis and inhibits gluconeogenesis

Key Enzymes & Blood Glucose Regulation

• Fructose-2,6-bisphosphate is a critical regulator
• High levels activate PFK-1 (glycolysis) and inhibit fructose-1,6-bisphosphatase (gluconeogenesis)
• Low levels do the opposite
• A bifunctional enzyme, PFK-2/FBPase-2, controls fructose-2,6-bisphosphate levels
• Regulated by insulin (stimulates glycolysis) and glucagon (stimulates gluconeogenesis)

Cori Cycle

• Transports lactate from muscles to the liver for glucose conversion
• Steps:
  • Anaerobic glycolysis in muscles generates lactate
  • Lactate enters the bloodstream and reaches the liver
  • The liver converts lactate back to glucose via gluconeogenesis
  • Glucose is released back into the bloodstream and taken up by muscles

Conditions for Pyruvate to Enter Aerobic Respiration

• Oxygen must be present, allowing Pyruvate to proceed to the citric acid cycle
• Pyruvate is transported into the mitochondrial matrix via the pyruvate translocase protein

Pyruvate Transport & Conversion to Acetyl-CoA

• Pyruvate translocase moves pyruvate into the mitochondrial matrix by transport
• Reaction Steps:
  • Decarboxylation occurs as pyruvate loses a CO₂ molecule
  • Oxidation occurs as the remaining 2-carbon molecule is oxidized, reducing NAD⁺ to NADH
  • Coenzyme A is added as the 2-carbon fragment attaches to Coenzyme A, forming Acetyl-CoA
• This process is due to the enzyme pyruvate dehydrogenase complex (PDC)
• The reaction produces Acetyl-CoA, CO₂, and NADH

Fates of Acetyl-CoA

• Acetyl-CoA enters the TCA cycle for ATP production
• Lipid synthesis occurs in high-energy conditions, using Acetyl-CoA for fatty acid synthesis

Pyruvate Dehydrogenase Complex (PDC) Components

• The enzymes involved include:
  • E1: Pyruvate dehydrogenase
  • E2: Dihydrolipoyl transacetylase
  • E3: Dihydrolipoyl dehydrogenase
• The catalytic coenzymes (not consumed) include thiamine pyrophosphate (TPP), lipoamide, and FAD
• Stoichiometric coenzymes (used up) include Coenzyme A and NAD⁺

Steps in Pyruvate to Acetyl-CoA Conversion

• Decarboxylation is when pyruvate is bound by E1, releasing CO₂
• Oxidation occurs as lipoamide on E2 transfers the remaining 2-carbon unit
• Acetyl-CoA formation occurs as the acetyl group is transferred to Coenzyme A by E2
• Regeneration of lipoamide occurs as E3 transfers electrons to FAD, then to NAD⁺

Rate-Limiting Step of Pyruvate Decarboxylation

• Pyruvate dehydrogenase (E1) carries this out
• This step commits pyruvate to aerobic metabolism

Oxidation Step in Pyruvate Conversion

• Starts with the Hydroxyethyl-TPP intermediate
• Ends with the Acetyl group bound to lipoamide
• Dihydrolipoyl transacetylase (E2) is what carries this out

Formation of Acetyl-CoA

• Acetyl-lipoamide is the substrate
• Acetyl-CoA is the product
• Dihydrolipoyl transacetylase (E2) is the enzyme

Regeneration of Lipoamide

• Enzyme: Dihydrolipoyl dehydrogenase (E3)
• FAD oxidizes lipoamide, then transfers electrons to NAD⁺
• Product: NADH

Pyruvate Dehydrogenase Regulation

• Allosteric regulation involves:
  • Inhibitors: ATP, NADH, Acetyl-CoA (high energy state)
  • Activators: ADP, Pyruvate (low energy state)
• Covalent regulation involves:
  • Pyruvate dehydrogenase kinase (PDK) phosphorylates and inactivates PDC
  • Pyruvate dehydrogenase phosphatase (PDP) dephosphorylates and activates PDC

Regulation by Metabolic Signals

• PDK Activation (inhibits PDC) by NADH, Acetyl-CoA, ATP
• PDK Inhibition (activates PDC) by ADP, Pyruvate
• PDP Activation (activates PDC) by Ca²⁺, and promotes energy production in active tissues