Glycolysis pt 2

What enzyme catalyzes Step 5 and what is its ΔG'°?

Triose phosphate isomerase converts DHAP to glyceraldehyde 3-phosphate (GAP). ΔG'° = +7.5 kJ/mol (thermodynamically unfavorable/reversible).

Which carbons of glucose end up in GAP after aldol cleavage?

DHAP contains carbons 1, 2, and 3 of glucose; GAP contains carbons 4, 5, and 6. After triose phosphate isomerase, both 3-carbon units proceed as GAP with mixed carbon origins.

What is Step 6 of glycolysis, which enzyme catalyzes it, and what is its ΔG'°?

Oxidation of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), incorporating inorganic phosphate and producing NADH. ΔG'° = +6.3 kJ/mol.

Why is Step 6 the first energy-yielding step of glycolysis?

It generates NADH from NAD⁺ and creates a high-energy acyl phosphate bond in 1,3-bisphosphoglycerate, enabling net ATP production in Step 7.

What is the role of the active-site cysteine in GAPDH?

It forms a high-energy covalent thioester intermediate with the substrate, which is then attacked by inorganic phosphate (phosphorolysis) to release 1,3-bisphosphoglycerate.

What are the 5 steps of the GAPDH mechanism?

(1) GAP binds and active-site Cys thiolate attacks the carbonyl; (2) a thiohemiacetal forms; (3) NAD⁺ oxidizes the intermediate to a thioester, producing NADH; (4) NADH leaves and is replaced by NAD⁺; (5) inorganic phosphate attacks the thioester, releasing 1,3-bisphosphoglycerate.

Why is GAPDH sensitive to oxidative stress?

Its active-site cysteine can be oxidized and inactivated under conditions of oxidative stress, inhibiting glycolysis.

What is Step 7 of glycolysis, which enzyme catalyzes it, and what is its ΔG'°?

Transfer of phosphate from 1,3-bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate by phosphoglycerate kinase. ΔG'° = -18.5 kJ/mol.

Why is Step 7 considered reversible despite being thermodynamically favorable?

It is coupled to the GAPDH reaction (Step 6), which is thermodynamically unfavorable — the two reactions together are near equilibrium, making Step 7 reversible in context.

What is Step 8 of glycolysis, which enzyme catalyzes it, and what is its ΔG'°?

Migration of the phosphoryl group from C3 to C2 of 3-phosphoglycerate to form 2-phosphoglycerate by phosphoglycerate mutase (requires Mg²⁺). ΔG'° = +4.4 kJ/mol.

What is the mechanism of phosphoglycerate mutase?

An active-site histidine (phosphohistidine) donates its phosphate to C2 of the substrate, forming a 2,3-bisphosphoglycerate intermediate; then the enzyme retrieves the phosphate from C3, yielding 2-phosphoglycerate and regenerating phosphohistidine.

What is Step 9 of glycolysis, which enzyme catalyzes it, and what is its ΔG'°?

Dehydration of 2-phosphoglycerate to phosphoenolpyruvate (PEP) by enolase, releasing water. ΔG'° = +7.5 kJ/mol.

Why is 2-phosphoglycerate not a good enough phosphate donor on its own?

Loss of phosphate from 2-PG would yield a secondary alcohol with no further stabilization; dehydration to PEP creates a high-energy enol phosphate that greatly increases the phosphate's transfer potential.

What is Step 10 of glycolysis, which enzyme catalyzes it, and what is its ΔG'°?

Transfer of phosphate from PEP to ADP to form pyruvate and ATP by pyruvate kinase (requires Mg²⁺ and K⁺). ΔG'° = -31.4 kJ/mol; highly favorable and irreversible.

How does pyruvate tautomerization drive ATP production in Step 10?

PEP loses its phosphate to form pyruvate in the unstable enol form, which spontaneously tautomerizes to the keto form. This tautomerization lowers the effective concentration of the product, driving the reaction strongly toward ATP formation.

How is pyruvate kinase (Step 10) regulated?

Inhibited by high ATP and regulated by divalent metals; the irreversibility under cellular conditions makes it a key regulatory point.

What is the overall balanced equation for glycolysis?

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

What are the three possible fates of pyruvate after glycolysis?

(1) Aerobic conditions: converted to acetyl-CoA and oxidized via the citric acid cycle; (2) Anaerobic/hypoxic in animals: reduced to lactate (lactic acid fermentation); (3) Anaerobic in yeast: converted to ethanol and CO₂ (alcoholic fermentation).

Why must NADH be reoxidized to NAD⁺ for glycolysis to continue?

Glycolysis requires NAD⁺ as an electron acceptor in Step 6; if NADH is not reoxidized, NAD⁺ is depleted and glycolysis halts.

What is the Warburg effect in cancer cells?

Tumor cells preferentially use glycolysis even under aerobic conditions (aerobic glycolysis), upregulating glucose transporters (GLUT1/GLUT3) and glycolytic enzymes via HIF-1, producing only 2 ATP/glucose instead of ~30 ATP from aerobic respiration.

What is HIF-1 and what is its role in cancer metabolism?

Hypoxia-inducible factor-1 (HIF-1) is activated under low-oxygen conditions in tumors; it upregulates glycolytic enzymes and glucose transporters, promoting the Warburg effect.

What is lactic acid fermentation and when does it occur?

Under anaerobic or hypoxic conditions, pyruvate is reduced to lactate by lactate dehydrogenase using NADH, regenerating NAD⁺. It occurs in vigorously contracting muscle, erythrocytes, and some microorganisms.

What is alcoholic fermentation and where does it occur?

Under anaerobic conditions in yeast, pyruvate is decarboxylated to acetaldehyde and then reduced to ethanol using NADH, regenerating NAD⁺.