Glycolysis Comprehensive Notes

Glycolysis

Learning Objectives

• Outline important steps, regulation, and clinical significance of Glycolysis.

Case Study: Blood Glucose Analysis

• A medical intern collected a blood sample in a plain vacutainer for blood glucose level analysis.

• The sample was kept for 8 hours before being sent for analysis.

• The blood glucose level was surprisingly low, but the patient showed no signs of hypoglycemia.

• Points for consideration:

  1. Possible errors in the process.

  2. Effects of prolonged sample storage.

  3. Reasons for decreased blood glucose levels during storage.

Definition of Glycolysis

• Glycolysis: Conversion of Glucose to pyruvate/lactate + ATP

Importance of Glycolysis

• Occurs in all cells.

• Main energy source for RBCs.

• Predominant energy pathway during strenuous exercise (anaerobic glycolysis).

• Provides carbon skeletons for non-essential amino acids and glycerol synthesis.

• Most steps are reversible and utilized in gluconeogenesis.

Key Roles of Glycolysis in RBCs

• Primary Energy Source (ATP Production)

  • RBCs rely entirely on anaerobic glycolysis for ATP production.

  • ATP is required for ion pump function ($Na^+/K^+-ATPase$), membrane integrity, and deformability, which facilitates passage through capillaries.

• Prevention of Hemolysis

  • ATP from glycolysis maintains the biconcave shape of RBCs and prevents premature destruction (hemolysis).

• 2,3-Bisphosphoglycerate (2,3-BPG) Production

  • The glycolytic intermediate 1,3-BPG is converted to 2,3-BPG, which regulates oxygen binding to hemoglobin.

  • Increased 2,3-BPG lowers hemoglobin's oxygen affinity, enhancing oxygen delivery to tissues.

• Reduction of Methemoglobin (HbM) via NADH

  • Glycolysis produces NADH, which is essential for the methemoglobin reductase enzyme.

  • This prevents the accumulation of methemoglobin (oxidized hemoglobin), which cannot bind oxygen.

• Pentose Phosphate Pathway (PPP) Support

  • Glycolysis provides precursors for the pentose phosphate pathway (PPP), which generates NADPH.

  • NADPH protects RBCs from oxidative damage by maintaining glutathione in its reduced form.

Glucose Entry into Cells

• GLUT4:

  • Found in muscle cells and adipocytes.

  • Insulin-dependent.

• GLUT2:

  • Found in liver cells and beta cells of the pancreas.

  • Insulin-independent.

Reactions of Glycolysis

• Site: All cells in the body.

• Subcellular Site: Cytosol fraction.

Steps of Glycolysis

• 10 reactions take place.

• 3 reactions are irreversible.

• Three phases:

  • Energy investment

  • Splitting

  • Energy generation

Reaction of Glycolysis - Enzymes and Conversions

• Step 1:

  • Enzymes: Hexokinase / Glucokinase

  • Reaction: Glucose → Glucose-6-phosphate

  • $ATP \rightarrow ADP + Mg^{2+}$

• Step 2:

  • Enzyme: Phosphohexose isomerase

  • Reaction: Glucose-6-phosphate → Fructose-6-phosphate

• Step 3:

  • Enzyme: Phosphofructokinase

  • Reaction: Fructose-6-phosphate → Fructose-1,6-bisphosphate

  • $ATP \rightarrow ADP + Mg^{2+}$

Glycolysis Reactions Continued

• Step 4:

  • Enzyme: Aldolase

  • Reaction: Fructose 1,6-bisphosphate → Dihydroacetone phosphate + Glyceraldehyde 3-phosphate

• Step 5:

  • Enzyme: Isomerase

  • Reaction: Dihydroacetone phosphate → Glyceraldehyde 3-phosphate

Glycolysis Reactions Continued 2

• Step 6:

  • Enzyme: Glyceraldehyde 3 phosphate dehydrogenase

  • Reaction: Glyceraldehyde 3-phosphate → 1,3-bisphosphoglycerate

  • $NAD^+ \rightarrow NADH + H^+$

• Step 7:

  • Enzyme: Phosphoglycerate Kinase

  • Reaction: 1,3 bisphosphoglycerate → 3-phosphoglycerate

  • $ADP \rightarrow ATP$

Glycolysis Reactions Continued 3

• Step 8:

  • Enzyme: Phosphoglycerate Mutase

  • Reaction: 3-phosphoglycerate → 2-phosphoglycerate

• Step 9:

  • Enzyme: Enolase

  • Reaction: 2-phosphoglycerate → Phosphoenolpyruvate

  • Removal of $H_2O$ and requires $Mg^{2+}$

Glycolysis Reactions Continued 4

• Step 10:

  • Enzyme: Pyruvate Kinase

  • Reaction: Phosphoenolpyruvate → Pyruvate

  • $ADP \rightarrow ATP + Mg^{2+}$

  • Total pyruvate formed is 2 molecules per glucose.

Anaerobic Conditions

• Enzyme: Lactate Dehydrogenase

• Reaction: Pyruvate → Lactate

• $NADH + H^+ \rightarrow NAD^+$

Interrelation between Glycolysis and Acetyl CoA Production.

• Under anaerobic conditions, the conversion of pyruvate to Acetyl CoA is blocked due to lack of oxygen.

• Glyceraldehyde-3-P is converted to 1,3-bisphosphoglycerate via Gly3PDH, producing NADH.

• Pyruvate is converted to Lactate via LDH, utilizing NADH.

Energetics of Glycolysis

• Calculate overall energy (ATP's and NADH + H+).

Regulation of Glycolysis

• Glucokinase

  • Has a high Km for glucose compared to Hexokinase.

  • Low affinity for glucose; acts when glucose is abundant.

  • Glucose-6-phosphate inhibits hexokinase.

Hexokinase vs Glucokinase

• Hexokinase has recently been shown to display very low affinity for fructose but no affinity for galactose.

Phosphofructokinase (PFK)

• Important rate-limiting enzyme.

• ATP and Citrate are allosteric inhibitors.

• AMP is an allosteric activator.

• Fructose-2,6-bisphosphate regulates PFK activity.

Regulation via Allosteric Inhibition

• The allosteric inhibitor is most effective when substrate concentration is low.

• When more substrate molecules are available, stringent regulation is less necessary.

• Fructose-6-phosphate + ATP → Fructose-1,6-bisphosphate + ADP, catalyzed by Phosphofructokinase 1.

Regulation of PFK1

• Committed step in glycolysis.

• ATP acts as an allosteric inhibitor (negative modifier) of PFK1.

• High ATP levels slow down glycolysis; ATP binds to the allosteric site, inhibiting the reaction.

• High AMP levels (indicating low ATP) act as an allosteric activator (positive modifier) of the enzyme.

Enolase Inhibition by Fluoride

• Fluoride removes magnesium and manganese ions, inhibiting the enzyme enolase.

• This consequently inhibits glycolysis.

Fructose-2,6-bisphosphate Regulation

• Fructose-6-phosphate can be converted to Fructose-2,6-bisphosphate by PFK-2, which activates PFK-1, thus promoting glycolysis.

• Alternatively, Fructose-2,6-bisphosphate can revert back to Fructose-6-phosphate.

Role of Fructose-2,6-Bisphosphate (F-2,6-BP)

• F-2,6-BP increases the activity of phosphofructokinase. It is formed from fructose-6-phosphate by PFK-2 (distinct from PFK-1).

• The activities of PFK2 and Fructose-2,6-bisphosphatase are reciprocally regulated.

Regulation of PFK2 and F-2,6-Bisphosphatase

• The two enzyme activities are present on the same polypeptide chain, a tandem enzyme.

• Addition of a phosphate group to the tandem enzyme activates F-2,6-bisphosphatase and inactivates PFK2, leading to a fall in F-2,6-bisphosphate, slowing down glycolysis.

• Dephosphorylation has the opposite effect.

• An increase in cyclic AMP level in the cell phosphorylates the enzymes.

Phosphofructokinase-2 (PFK-2) / Fructose Bisphosphatase-2 (FBPase-2)

• An enzyme indirectly responsible for regulating the rates of glycolysis and gluconeogenesis in cells.

• It catalyzes formation and degradation of a significant allosteric regulator, fructose-2,6-bisphosphate (Fru-2,6-P2) from substrate fructose-6-phosphate.

• Fru-2,6-P2 contributes to the rate-determining step of glycolysis as it activates enzyme phosphofructokinase 1 in the glycolysis pathway and inhibits fructose-1,6-bisphosphatase 1 in gluconeogenesis.

Regulation of Pyruvate Kinase

• Insulin activates pyruvate kinase, favoring glycolysis.

• ATP, Glucagon, and Glucocorticoids inhibit pyruvate kinase.

Rapaport Leubering Cycle (BPG Shunt)

• In erythrocytes, this cycle bypasses one step of glycolysis.

• Bisphosphoglycerate mutase converts 1,3-bisphosphoglycerate (BPG) to 2,3-BPG.

• BPG-phosphatase then removes the phosphate group to form 3-phosphoglycerate.

Significance of BPG

1. 2,3-BPG binds to hemoglobin, reducing its oxygen affinity, facilitating oxygen unloading in tissues.

2. Under hypoxic conditions, 2,3-BPG concentration in RBCs increases, favoring oxygen release to tissues even at low $pO_2$.

3. The compensatory increase in 2,3-BPG at high altitudes favors oxygen dissociation. BPG is increased in fetal circulation.

4. In this shunt pathway, no ATP is generated.

Reference

• Textbook of Biochemistry by DM Vasudevan, 8th edition.