3. Glycolysis and Pyruvate Metabolism

Where does glycolysis occur?

In the cytoplasm of aerobic and anaerobic organisms.

What is the purpose of glycolysis?

It is a key ATP-producing pathway that converts glucose into pyruvate.

What are the two phases of glycolysis?

1. Energy Investment (steps 1–5, consumes ATP). consumes 2 ATP and converts glucose to two glyceraldehyde-3-P

2. Energy Payoff (steps 6–10, generates ATP and NADH). produces 4 ATP and two pyruvates

How many ATP are consumed in the investment phase?

2 ATP.

What is produced during the payoff phase?

4 ATP (substrate-level phosphorylation), 2 NADH, and 2 pyruvate.

What is the NET yield of glycolysis per glucose?

2 ATP, 2 NADH, and 2 pyruvate.

What intermediate is produced at the “split” of glucose?

Two molecules of glyceraldehyde-3-phosphate (G3P).

What does hexokinase do in step 1?

Phosphorylates glucose → glucose-6-phosphate, trapping it in the cell.

Is the hexokinase reaction reversible?

No—step 1 is irreversible under cellular conditions.

How is hexokinase regulated?

Inhibited by its product, glucose-6-phosphate.

What is substrate-level phosphorylation?

Direct transfer of a phosphate from a high-energy intermediate to ADP to form ATP.

Which glycolysis steps use substrate-level phosphorylation?

Step 7 (phosphoglycerate kinase) and Step 10 (pyruvate kinase).

What is the committed step of glycolysis?

PFK-1 converting fructose-6-phosphate → fructose-1,6-bisphosphate.

What inhibits PFK-1?

ATP and citrate.

What activates PFK-1?

AMP and fructose-2,6-bisphosphate.

What activates pyruvate kinase?

Fructose-1,6-bisphosphate and AMP.

What inhibits pyruvate kinase?

ATP and acetyl-CoA.

What is the product of step 1?

Glucose-6-phosphate (G6P).

Which enzyme converts G6P → fructose-6-phosphate?

Phosphoglucose isomerase (Step 2).

What does aldolase produce?

DHAP and G3P (step 4).

What enzyme interconverts DHAP ↔ G3P?

Triose phosphate isomerase (Step 5).

What does G3P dehydrogenase generate?

1,3-BPG and NADH.

What is the high-energy molecule just before ATP formation in step 10?

Phosphoenolpyruvate (PEP).

What enzyme converts pyruvate → lactate under anaerobic conditions?

Lactate dehydrogenase (LDH).

Why is LDH important?

It regenerates NAD⁺ so glycolysis can continue.

What does the pyruvate dehydrogenase complex (PDC) do?

Converts pyruvate → acetyl-CoA via oxidative decarboxylation.

How many enzymes make up PDC?

Three enzymes: E1, E2, and E3.

Which vitamin is required for optimal PDC function?

Thiamine (B1), through its coenzyme TPP.

What happens to PDC activity during thiamine deficiency?

It decreases, impairing acetyl-CoA production.

Step 1: Hexokinase reaction Enzyme & product?

Hexokinase → G6P (irreversible).

Step 2: Phosphoglucose Isomerase (aka Phosphoglucoisomerase) Reaction Enzyme & product?

Phosphoglucose isomerase → F6P.

Step 3: Phosphofructokinase-1 (PFK-1) Reaction Enzyme & product?

PFK-1 → F1,6BP (committed step, irreversible).

Step 4: Enzyme & products? Aldolase Reaction

Aldolase → DHAP + G3P.

Step 5: Triose Phosphate Isomerase Reaction Enzyme?

Triose phosphate isomerase converts DHAP ↔ G3P.

Step 6: Glyceraldehyde-3-Phosphate Dehydrogenase Reaction. Enzyme & product?

G3P dehydrogenase → 1,3-BPG + NADH.

Phosphoglycerate Kinase Reaction Step 7: Enzyme & product?

Phosphoglycerate kinase → 3-PG + ATP. (First substrate-level phosphorylation.)

Step 8: Phosphoglycerate Mutase Reaction Enzyme & product?

Phosphoglycerate mutase → 2-PG.

Step 9: Enolase Enzyme & product?

Enolase → PEP + H₂O.

Step 10: Pyruvate Kinase Reaction Enzyme & product?

Pyruvate kinase → Pyruvate + ATP (irreversible).

What are the final products of glycolysis?

2 pyruvate, 2 ATP (net), and 2 NADH.

Why does phosphorylation trap glucose inside the cell?

Because glucose-6-phosphate cannot cross the plasma membrane.

What is the Km of hexokinase for glucose?

0.1 mM, meaning hexokinase has very high affinity for glucose.

What is the typical intracellular glucose concentration?

~4 mM, which is far above hexokinase’s Km.

How is hexokinase regulated?

Allosterically inhibited by its product, glucose-6-phosphate (G6P).

What is Km? What does a low Km value indicate?

Km is the substrate concentration at which an enzyme reaches half of its maximum velocity (½ Vmax). High affinity between an enzyme and its substrate.

Why does hexokinase have high affinity for glucose?

Because it has a low Km, meaning it reaches half-maximal activity at very low glucose concentrations.

What is Hexokinase I and where is it found?

Isoform in brain and red blood cells, strongly inhibited by G6P and has very high glucose affinity.

What is Hexokinase II and where is it found?

Isoform in muscle and adipose tissue, inhibited by G6P and insulin-responsive.

What is Hexokinase III and where is it found?

Isoform found in various tissues, regulated similarly to I and II but less well characterized.

What is Hexokinase IV and where is it found?

Isoform in liver and pancreatic β-cells, not inhibited by G6P and regulated by GKRP and insulin

What role does glucokinase play in pancreatic β-cells?

It acts as a glucose sensor, detecting elevated blood glucose and triggering insulin secretion.

What happens to intracellular glucose levels in β-cells after a meal?

Glucose enters β-cells via transporters, raising intracellular glucose to millimolar levels.

How does glucokinase-driven glycolysis help initiate insulin release?

It increases the ATP/ADP ratio, which closes ATP-sensitive K⁺ channels.

What is the consequence of K⁺ channel closure in β-cells?

Membrane depolarization opens Ca²⁺ channels, allowing calcium influx.

What does Ca²⁺ influx trigger in β-cells?

Exocytosis of insulin-containing vesicles.

What does the released insulin do in other tissues?

It promotes glucose uptake and energy production in insulin-sensitive tissues (muscle, adipocytes, heart, etc.).

How is the muscle isoform of pyruvate kinase (PK-M1) regulated?

PK-M1 in muscle is not hormonally regulated and simply responds to energy demand.

What regulates the liver isoform of pyruvate kinase (PK-L)?

PK-L is activated by fructose-1,6-bisphosphate and inhibited by ATP and alanine; hormonally, glucagon inhibits via PKA-mediated phosphorylation and insulin activates via dephosphorylation.

What is unique about pyruvate kinase regulation in the brain (PK-M1)?

Brain PK-M1 has minimal hormonal regulation and maintains high constitutive activity to support constant energy needs.

What regulates the red blood cell isoform of pyruvate kinase (PK-R)?

PK-R is activated by fructose-1,6-bisphosphate and inhibited by ATP, and its activity is tied to oxygen demand and glycolytic flux.

How is the pancreatic β-cell isoform of pyruvate kinase (PK-M2) regulated?

PK-M2 is activated by fructose-1,6-bisphosphate, inhibited by ATP, and is indirectly influenced by glucose metabolism and insulin signaling.

What are the two general pathways for recycling NADH?

NADH can be recycled through aerobic or anaerobic pathways.

What does NADH represent in metabolism?

NADH represents stored energy that can be used to make ATP.

hat happens to NADH under aerobic conditions?

NADH is oxidized in the electron transport chain, producing ATP through oxidative phosphorylation.

What are the three key regulatory enzymes of glycolysis? and why

Hexokinase (step 1), PFK-1 (step 3), and pyruvate kinase (step 10). Because they are irreversible, highly exergonic steps catalyzed by enzymes whose activity can be modulated to control pathway flux.

How is glycolysis regulated by energy charge?

ATP inhibits glycolysis, while AMP activates it.

What hormone promotes and inhibits glycolysis?

Insulin promotes, glucagon inhibits

What is tautomerization?

A type of isomerization where a molecule rapidly interconverts between two structural forms called tautomers.

What intermediate is formed when PEP transfers its phosphate to ADP?

Enol-pyruvate, the enol form of pyruvate.

What happens to enol-pyruvate after phosphate transfer?

It undergoes spontaneous tautomerization to the more stable keto pyruvate.

Why does PEP hydrolysis have such a large negative ΔG°′?

Because the enol → keto tautomerization of pyruvate is highly favorable and drives the reaction forward.

Why does tautomerization make PEP a high-energy molecule?

The spontaneous conversion of enol-pyruvate to keto-pyruvate “locks in” the products, making PEP → pyruvate + ATP strongly exergonic.

Is an enzyme required for the enol → keto conversion of pyruvate?

No — the tautomerization occurs spontaneously in solution.

How is NAD⁺ regenerated during anaerobic glycolysis?

NAD⁺ is regenerated when NADH is oxidized by either lactate dehydrogenase (forming lactate) or alcohol dehydrogenase (forming ethanol via acetaldehyde), allowing glycolysis to continue without oxygen ANAEROBIC

What happens to pyruvate under aerobic conditions?

Pyruvate is converted to acetyl-CoA, which enters the TCA cycle.

What happens to pyruvate under anaerobic conditions?

Pyruvate is converted to lactate (in animals) or ethanol + CO₂ (in yeast)