Glycolysis, gluconeogenesis, metabolic regulation | Principles of bioenergetics and fuel molecule metabolism | Bio/biochem

Glycolysis, gluconeogenesis, and the pentose phosphate pathway

Metabolism encompasses the full range of chemical processes in living organisms, divided into two complementary aspects: catabolism (breaking down molecules to release energy) and anabolism (using energy to build and store complex molecules).

In aerobic metabolism, oxygen is required to completely oxidize a source of fuel—commonly glucose—into carbon dioxide and water while capturing released energy in the form of ATP. Under these conditions, one molecule of glucose typically yields around 30 ATP. The overall reaction is often summarized as:

$C_{6} H_{12} O_{6} + 6 O_{2} \to 6 C O_{2} + 6 H_{2} O$

Glucose ( $C_{6} H_{12} O_{6}$ ), obtained from the diet, provides carbons and hydrogens.
$O_{2}$ (molecular oxygen) becomes the final electron acceptor in the electron transport chain, ultimately forming water.
$C O_{2}$ emerges when the carbons (and original oxygens) from glucose are released in processes like the Krebs cycle.
$H_{2} O$ arises during the final stages of the electron transport chain, as electrons and protons combine with the inhaled $O_{2}$ .

Glycolysis

Glycolysis is the central metabolic pathway in which glucose is broken down into pyruvate, yielding small amounts of ATP and NADH.

Under aerobic conditions, pyruvate enters the mitochondria to fuel further energy production via the citric acid cycle and oxidative phosphorylation.

Steps in glycolysis:

Hexokinase phosphorylation
Glucose ( $C_{6} H_{12} O_{6}$ ) enters the cell and is phosphorylated by hexokinase, using an ATP to form glucose-6-phosphate. This modification traps glucose within the cell and makes it more reactive.
Isomerization
An isomerase converts glucose-6-phosphate into fructose-6-phosphate, preparing the molecule for further phosphorylation and eventually split into two three‑carbon units.
Second Phosphorylation
Phosphofructokinase phosphorylates fructose-6-phosphate (using another ATP), producing fructose‑1,6-bisphosphate. This enzyme is a key control point in glycolysis, responding to cellular ATP/ADP levels.
Sugar Cleavage
Aldolase splits fructose-1,6-bisphosphate into two three-carbon molecules: dihydroxyacetone phosphate (DHAP) and glyceraldehyde‑3‑phosphate (G3P).
Isomerization to G3P
An isomerase converts DHAP into glyceraldehyde‑3‑phosphate, so the pathway continues with two G3P molecules. Up to this point, two ATPs have been spent per glucose.
Oxidation and Phosphate Addition
Each G3P is oxidized, generating NADH, and is phosphorylated again to form 1,3-bisphosphoglycerate—this does not require an additional ATP.
First ATP Generation
1,3-bisphosphoglycerate donates a high-energy phosphate to ADP, producing ATP (via substrate-level phosphorylation) and 3-phosphoglycerate.
Phosphate Rearrangement
A mutase enzyme shifts the phosphate group from carbon 3 to carbon 2, forming 2-phosphoglycerate.
Dehydration
Enolase removes water, converting 2-phosphoglycerate into phosphoenolpyruvate (PEP). This reaction heightens the phosphate group’s energy.
Second ATP Formation
Pyruvate kinase transfers the phosphate from PEP to ADP, yielding another ATP and producing pyruvate, which can be further processed aerobically (in mitochondria) or anaerobically (fermentation).

First half of glycolysis showing energy-requiring steps and ATP investment in phosphorylation

Second half of glycolysis showing energy-releasing steps producing NADH and ATP molecules

Anaerobic metabolism

However, glycolysis also serves as the endpoint for anaerobic metabolism, or fermentation. Fermentation starts with partial oxidation of glucose to pyruvate, with 2 net ATP produced per glucose. Then, pyruvate is converted into lactate (in animals), called lactic acid fermentation, or ethanol and carbon dioxide (in yeast), called alcohol fermentation, to regenerate NAD⁺.

Carbohydrates stored as glycogen in animals or starch in plants can be rapidly mobilized via feeder pathways, providing additional glucose for glycolysis.

Gluconeogenesis

Gluconeogenesis is essentially the reverse process of glycolysis. It generates glucose from non-carbohydrate precursors such as lactate, amino acids, and glycerol. This pathway is vital during fasting or intense exercise, ensuring a continuous supply of glucose to critical tissues like the brain.

The pathway begins with the conversion of pyruvate into oxaloacetate via pyruvate carboxylase, an enzyme that requires ATP and biotin as a cofactor. Oxaloacetate is then decarboxylated and phosphorylated to form phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase (PEPCK), using GTP as an energy source.

Subsequent reactions reverse most of the glycolytic pathway through a series of enzyme-catalyzed steps until fructose-1,6-bisphosphate is formed. This intermediate is then hydrolyzed by fructose-1,6-bisphosphatase, a key regulatory enzyme in gluconeogenesis, which removes a phosphate group to yield fructose-6-phosphate.

The pathway continues until glucose-6-phosphate is generated, which is then dephosphorylated by glucose-6-phosphatase to produce free glucose that can be released into the bloodstream.

Gluconeogenesis is tightly regulated at both the transcriptional and allosteric levels, with hormones such as glucagon and cortisol upregulating the pathway during periods of low blood sugar, while insulin inhibits it when glucose is abundant.

Glycolysis and gluconeogenesis intermediates and enzymes pathway diagram

The pentose phosphate pathway (PPP) operates parallel to glycolysis and fulfills two key functions:

it produces NADPH, which is crucial for reductive biosynthesis and maintaining cellular redox balance
it generates ribose-5-phosphate for nucleotide synthesis. By diverting glucose-6-phosphate from glycolysis, the PPP provides the cell with both energy and the building blocks necessary for DNA, RNA, and various biosynthetic processes.

The net molecular and energetic outcomes of these respiration processes are tightly coordinated through intricate metabolic regulation. Cells maintain a dynamic steady state by controlling pathway fluxes through mechanisms such as allosteric regulation, hormonal signaling, and genetic control. For example, key enzymes in glycolysis and gluconeogenesis are regulated in response to energy levels, ensuring that when one pathway is active, the other is suppressed. Similarly, the metabolism of glycogen is finely tuned by enzymes that control both its synthesis and breakdown, allowing organisms to store energy when abundant and mobilize it when needed.

Principles of metabolic regulation

Metabolic pathways are typically governed by rate-limiting steps, where the slowest or earliest irreversible reactions serve as primary control points. Regulation may be positive (amplifying a process) or negative (suppressing it), often forming feedback loops that help maintain a dynamic steady state (homeostasis) in living organisms.

Glycolysis, which converts glucose into pyruvate, is driven by crucial enzymes such as hexokinase, phosphofructokinase, and pyruvate kinase. When the cell has abundant ATP, glycolysis slows, favoring gluconeogenesis instead—essentially a reversal of glycolysis that synthesizes glucose. Conversely, high ADP levels (and low ATP) stimulate glycolysis. Hormones like epinephrine trigger muscle glycolysis and increase blood glucose, while fructose-2,6-bisphosphate promotes glycolysis and downregulates gluconeogenesis.

Glycogen metabolism highlights another layer of regulation. Glycogen breakdown yields glucose-1-phosphate, which can be channeled into multiple pathways (e.g., glycolysis, pentose phosphate pathway) after converting to glucose-6-phosphate. Hormones like glucagon and epinephrine can initiate a cAMP cascade that enhances glycogen phosphorylase activity (breaking down glycogen) and inhibits glycogen synthase (building glycogen). Conversely, lowered cAMP levels shift the balance to glycogen synthesis.