Glycolysis and Cellular Respiration

• Organic Chemistry II is presented by Md Golam Moula, Ph.D.

Glycolysis

• Glycolysis involves breaking down glucose into pyruvate.
• This intracellular process occurs in the cytoplasm.

Types of Glycolysis:

• Aerobic Glycolysis: Glucose is converted to pyruvate and then to acetyl CoA, followed by the TCA cycle and oxidative phosphorylation.
• Anaerobic Glycolysis: Glucose is converted to pyruvate and then to lactate.
• Red blood cells (RBCs) use anaerobic glycolysis because they do not have mitochondria.

Glycolysis Overview

• Glycolysis converts glucose into 2 pyruvic acid molecules + 2 ATP + 2 NADH.
• This process is divided into an energy investment phase and an energy harvest phase.
• The energy investment phase requires 2 ATP. The energy harvest phase yields 2 ATP and 2 NADH.

Energy Investment and Harvest Phase

• The initial steps require an investment of energy, using ATP.
• Subsequent steps harvest energy in the form of ATP and NADH.
• Two molecules of 3-carbon pyruvate are produced during the energy harvest phase.

Glycolysis Reaction Steps

• Glycolysis is a series of enzyme-catalyzed reactions that convert glucose into pyruvate.
• The process involves 10 steps:

1. Hexokinase

• Glucose + ATP → glucose-6-P + ADP

2. Phosphoglucose Isomerase

• Glucose-6-P (aldose) ↔ fructose-6-P (ketose)

3. Phosphofructokinase

• Fructose-6-P + ATP → fructose-1,6-bisP + ADP

4. Aldolase

• Fructose-1,6-bisphosphate ↔ dihydroxyacetone-P + glyceraldehyde-3-P

5. Triose Phosphate Isomerase (TIM)

• Dihydroxyacetone-P ↔ glyceraldehyde-3-P

6. Glyceraldehyde-3-phosphate Dehydrogenase

• Glyceraldehyde-3-P + NAD+ + Pi ↔ 1,3-bisphosphoglycerate + NADH + H+

7. Phosphoglycerate Kinase

• 1,3-bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP

8. Phosphoglycerate Mutase

• 3-phosphoglycerate ↔ 2-phosphoglycerate

9. Enolase

• 2-phosphoglycerate ↔ phosphoenolpyruvate + H₂O

10. Pyruvate Kinase

• Phosphoenolpyruvate + ADP → pyruvate + ATP

Glycolysis Location & Initial Steps

• Glycolysis happens in the cytosol of cells.
• Glucose begins glycolysis by turning into glucose-6-phosphate.
• The start of glycolysis requires energy, using two ~P bonds from ATP.

Hexokinase Mechanism

• Hexokinase catalyzes the reaction: Glucose + ATP → glucose-6-P + ADP
• The nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP occurs.
• ATP binds to the enzyme as a complex with Mg++.

Phosphoglucose Isomerase Mechanism

• Phosphoglucose Isomerase catalyzes the reaction: glucose-6-P (aldose) ↔ fructose-6-P (ketose)
• The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure.

Phosphofructokinase Mechanism

• Phosphofructokinase catalyzes the reaction: fructose-6-P + ATP → fructose-1,6-bisP + ADP
• This spontaneous reaction has a similar mechanism to that of Hexokinase.
• The Phosphofructokinase reaction is the rate-limiting step of Glycolysis.
• This enzyme is highly regulated.

Aldolase Mechanism

• Aldolase catalyzes: fructose-1,6-bisphosphate ↔ dihydroxyacetone-P + glyceraldehyde-3-P
• This reaction is an aldol cleavage, the reverse of an aldol condensation.
• A lysine residue at the active site functions in catalysis.
• The keto group of fructose-1,6-bisphosphate reacts with the amino group of the active site lysine, to make protonated Schiff base intermediate.
• Cleavage occurs at the bond between C3 & C4.

Triose Phosphate Isomerase (TIM)

• Triose Phosphate Isomerase catalyzes: dihydroxyacetone-P ↔ glyceraldehyde-3-P
• Glycolysis continues from glyceraldehyde-3-P.
• TIM's Keq favors dihydroxyacetone-P.
• The removal of glyceraldehyde-3-P by a rapid subsequent reaction allows throughput

Triosephosphate Isomerase Mechanism

• The ketose/aldose conversion uses acid/base catalysis.
• The conversion proceeds via an enediol intermediate.
• Active site Glu and His residues extract and donate protons during catalysis.

Glyceraldehyde-3-phosphate Dehydrogenase Mechanism

• Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi ↔ 1,3-bisphosphoglycerate + NADH + H+
• The aldehyde in glyceraldehyde-3-phosphate is oxidized into a carboxylic acid.
• This process drives an acyl phosphate formation with a high-energy bond (~P).
• This step is the only one in Glycolysis in which NAD+ becomes NADH.

Phosphoglycerate Kinase Mechanism

• Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP
• This phosphate transfer is reversible because the free energy is low.
• One ~P bond is cleaved and another is synthesized.
• The enzyme changes shape due to the substrate.
• Similar to that of Hexokinase.

Phosphoglycerate Mutase Mechanism

• Phosphoglycerate Mutase catalyzies: 3-phosphoglycerate ↔ 2-phosphoglycerate
• Phosphate moves position from the OH on C3 to the OH on C2.

Enolase Mechanism

• Enolase catalyzes: 2-phosphoglycerate ↔ phosphoenolpyruvate + H₂O
• This dehydration reaction is Mg++-dependent.
• Two Mg++ ions interact with oxygen atoms of the carboxyl group at the active site.
• Magnesium ions help stabilize the enolate anion intermediate that forms after lysine extracts H+ from C#2.

Pyruvate Kinase

Catalyzes: phosphoenolpyruvate + ADP → pyruvate + ATP

• This phosphate movement from PEP to ADP occurs spontaneously.
• PEP has a larger free energy of phosphate hydrolysis than ATP.
• Removal of phosphate from PEP yields an unstable enol.
• The enol spontaneously converts to the keto form of pyruvate.
• Potassium and magnesium inorganic cations bind to anionic residues at the active site of Pyruvate Kinase.

Glucose Phosphorylation

• Phosphorylating glucose with inorganic phosphate requires 3.3 kcal/mol.
• The reaction couples with one that releases energy.
• ATP is hydrolysed, releasing energy: ΔG° = -7.3 kcal/mol.
• glucose + ATP-> glucose 6P + ADP
• The net energy is a release: ΔG° = -4 kcal/mol.

Energy Yield of Glycolysis

• Aerobic Glycolysis:
  • Glucose to pyruvate yields 2 ATP/mol of glucose + 2 NADH
• Anaerobic Glycolysis:
  • Glucose to lactate yields 2 ATP/mol of glucose
  • Process that occurs in red blood cells, exercising muscles, white blood cells, kidney medulla, and eye.

Irreversible Steps in Glycolysis

• There are three irreversible steps in glycolysis that are not reversed by gluconeogenesis
  • Glucose to glucose 6-phosphate
  • Fructose 6-phosphate to fructose 1,6-bisphosphate
  • Phosphoenolpyruvate to pyruvate

Regeneration of NAD+

• NAD+ availability is necessary to continue of glycolysis
• In anaerobic conditions, electrons are transferred from NADH to pyruvate by lactate dehydrogenase, which forms NAD+ and lactate

Anaerobic Glycolysis

• RBCs lack mitochondria, and entirely depend entirely on anaerobic glycolysis for energy needs.
• In hypoxic conditions, muscles are partially supplied by anaerobic glycolysis.
• Anaerobic glycolysis is limited by Lactate build-up.
• pH is decreased due to Accumulation of lactic acid, thus it interferes with the function of the contractile machinery of the muscle.
• Elevated muscle lactate accounts for fatigue and pain.

Transport of Reducing Equivalents

• The inner mitochondrial membrane is impermeable to NADH.
• No transport protein can translocate NADH across this membrane directly
• NADH formed in glycolysis enters mitochondria for oxidation through these shuttles:
  • Glycerol phosphate (glycerol 3-P) shuttle
  • Malate-aspartate shuttle

Glycerol 3-Phosphate Shuttle

• Involves mitochondrial glycerol 3-phosphate dehydrogenase
• Reduces dihydroxyacetone phosphate to glycerol 3-phosphate (reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase)
• The reverse reaction is catalyzed by integral membrane flavoprotein (FAD) so electrons are transferred to CoQ of the electron transport chain.
• If used it leads to creation of 1.5 ATP instead of 2.5 ATP (1 NADH)
• Numbers of ATP created differ depending on
• In total 5 ATP for glycerol phosphate shuttle and 7 ATP for malate.
• This process is irreversible because of loss of energy.
• Involves cytosolic glycerol 3-phosphate dehydrogenase

Malate-Aspartate Shuttle

• Re-oxidation of malate in the mitochondrial matrix generates NADH that can pass electrons for 2.5 ATP.
• Completion of the shuttle cycle requires the activities of mitochondrial and cytosolic aspartate transaminase.
• Process driven by accumulation of NADH in the cytosol as well as NADH usage in the mitochodria.
• No energy is required.

Explore the crucial processes of glycolysis and cellular respiration, including the roles of the glycerol 3-phosphate and malate-aspartate shuttles in energy production. Understand the differences in ATP generation and the importance of anaerobic glycolysis in red blood cells and during strenuous exercise.