16. Glycolysis

Step 1: Hexokinase

Glucose is trapped inside the cell by phosphorylation (G6P cannot cross the plasma membrane). This is an irreversible, regulated step. Hexokinase is inhibited by its product, glucose-6-phosphate (product inhibition). In the liver, the isozyme glucokinase (hexokinase IV) has a higher $K_m$ for glucose (~10 mM vs 0.1 mM), allowing it to act as a glucose sensor.

Step 2: Phosphoglucose Isomerase

Aldose-to-ketose isomerization. The reaction is freely reversible and near equilibrium in the cell. The conversion to fructose-6-phosphate prepares the sugar for the second phosphorylation by PFK-1.

Step 3: Phosphofructokinase-1 (PFK-1)

This is the committed step and the most important regulatory point of glycolysis. PFK-1 is a tetrameric allosteric enzyme with complex regulation. It is activated by AMP (signals low energy charge) and fructose-2,6-bisphosphate (F2,6BP, the most potent activator). It is inhibited by ATP (high energy charge) and citrate (signals abundant TCA cycle intermediates). The irreversible commitment of F6P to glycolysis occurs here.

Step 4: Aldolase

Aldol cleavage splits the 6-carbon sugar into two 3-carbon fragments: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). Despite the highly positive $\Delta G^{\circ'}$, the reaction proceeds forward in vivo because the products are rapidly consumed by subsequent steps, keeping their concentrations very low ($Q \ll K_{eq}$).

Step 5: Triose Phosphate Isomerase (TPI)

TPI interconverts the two triose phosphates. Only G3P continues in glycolysis, so this reaction ensures both halves of glucose enter the payoff phase. TPI is often cited as a kinetically perfect enzyme — its catalytic efficiency ($k_{\text{cat}}/K_m \approx 4 \times 10^8 \text{ M}^{-1}\text{s}^{-1}$) approaches the diffusion-controlled limit, meaning every encounter between enzyme and substrate leads to product formation.

Step 6: Glyceraldehyde-3-Phosphate Dehydrogenase

This is the only oxidation step in glycolysis. The aldehyde group of G3P is oxidized to a carboxyl group, and NAD⁺ is reduced to NADH. The energy of oxidation is conserved by coupling it to the formation of a high-energy acyl phosphate bond in 1,3-bisphosphoglycerate (1,3-BPG). A crucial cysteine residue in the active site forms a thioester intermediate with the substrate.

Step 7: Phosphoglycerate Kinase

This is the first of two substrate-level phosphorylation steps. The high-energy acyl phosphate of 1,3-BPG ($\Delta G^{\circ'}_{\text{hydrolysis}} = -49.4$ kJ/mol) transfers its phosphoryl group to ADP, generating ATP. Steps 6 and 7 together constitute a coupled process: the oxidation energy captured in step 6 is used to drive ATP synthesis in step 7.

Step 8: Phosphoglycerate Mutase

The phosphoryl group is shifted from C-3 to C-2 through a 2,3-bisphosphoglycerate intermediate. This repositioning is necessary for the next step, where dehydration will create the high-energy enol phosphate of PEP.

Step 9: Enolase

Dehydration of 2-PG creates phosphoenolpyruvate (PEP), which has the highest phosphoryl transfer potential of any common metabolite ($\Delta G^{\circ'}_{\text{hydrolysis}} = -61.9$ kJ/mol). The removal of water traps the molecule in its unstable enol form, creating an enormous thermodynamic driving force for the next step. Enolase requires Mg$^{2+}$ ions and is inhibited by fluoride (through formation of a fluorophosphate complex with Mg$^{2+}$).

Step 10: Pyruvate Kinase

The second substrate-level phosphorylation. PEP transfers its phosphoryl group to ADP, yielding ATP and the enol form of pyruvate, which spontaneously tautomerizes to the much more stable keto form. This tautomerization is irreversible and drives the overall reaction strongly forward. Pyruvate kinase is the third regulated enzyme: it is activated by fructose-1,6-bisphosphate (feedforward activation) and inhibited allosterically by ATP and alanine. In the liver, glucagon promotes phosphorylation and inactivation of pyruvate kinase.

1. Aerobic Conditions: Oxidative Decarboxylation

In the presence of oxygen, pyruvate enters the mitochondrial matrix where the pyruvate dehydrogenase complex (PDH) catalyzes its irreversible oxidative decarboxylation to acetyl-CoA:

Acetyl-CoA then enters the citric acid cycle (TCA cycle) for complete oxidation, yielding additional NADH and FADH$_2$ that feed into the electron transport chain.

2. Anaerobic Conditions: Lactate Fermentation

When oxygen is limiting (e.g., during intense muscle exercise), pyruvate is reduced to lactate by lactate dehydrogenase (LDH):

This reaction serves the critical purpose of regenerating NAD⁺, which is essential for step 6 of glycolysis (G3P dehydrogenase) to continue. Without NAD⁺ regeneration, glycolysis would halt and ATP production would cease. Erythrocytes, which lack mitochondria, rely entirely on this anaerobic pathway. Lactate produced in muscle is transported to the liver via the Cori cycle, where it is converted back to glucose by gluconeogenesis.

3. Alcoholic Fermentation (Yeast)

In yeast and some other microorganisms, pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase (requires thiamine pyrophosphate, TPP), then reduced to ethanol by alcohol dehydrogenase:

Like lactate fermentation, this pathway regenerates NAD⁺ for continued glycolysis. This process is the basis of brewing and winemaking. The CO$_2$ produced is responsible for the carbonation in beer and the rising of bread dough.

1. Glycolysis is a 10-step cytoplasmic pathway that converts one glucose (C$_6$) into two pyruvate (C$_3$) molecules, yielding a net of 2 ATP + 2 NADH per glucose.

2. The pathway has two phases: the energy investment phase (steps 1–5, consuming 2 ATP) and the energy payoff phase (steps 6–10, producing 4 ATP + 2 NADH).

3. Three irreversible steps serve as control points: hexokinase (step 1), PFK-1 (step 3, the committed step), and pyruvate kinase (step 10). PFK-1 is the primary regulatory enzyme.

4. PFK-1 integrates multiple metabolic signals: it is activated by AMP and F2,6BP (low energy / fed state signals) and inhibited by ATP and citrate (high energy / ample fuel signals).

5. Pyruvate has three major fates: (a) aerobic oxidation to acetyl-CoA by the PDH complex, (b) reduction to lactate (regenerates NAD⁺ under anaerobic conditions), or (c) alcoholic fermentation to ethanol + CO$_2$ in yeast.

6. Substrate-level phosphorylation (steps 7 and 10) produces ATP directly by transfer of a phosphoryl group from a high-energy substrate to ADP, distinct from oxidative phosphorylation in mitochondria.

7. The overall $\Delta G^{\circ'}$ of glycolysis is −85 kJ/mol. Glycolysis alone captures a small fraction of glucose's total free energy; the majority is released in the TCA cycle and electron transport chain.