Glycolysis — Steps, Irreversible Enzymes, Energetics, Regulation and Clinical Links

Glycolysis — also called the Embden–Meyerhof pathway — is the oxidation of glucose to two molecules of pyruvate. It happens in the cytosol of every cell and does not need oxygen, so it is both the first stage of aerobic respiration and the only ATP source when oxygen or mitochondria are missing.

What happens to pyruvate depends on the cell. With oxygen and mitochondria, pyruvate enters the mitochondria, is converted to acetyl-CoA and feeds the citric acid cycle. Without them, lactate dehydrogenase reduces pyruvate to lactate — a step whose real purpose is to regenerate NAD+, without which the glyceraldehyde-3-phosphate dehydrogenase step would stop.

The pathway is taught in two halves. In the investment (preparatory) phase (steps 1–5) two ATP are spent to phosphorylate glucose and split it into two triose phosphates. In the payoff phase (steps 6–10) each of the two three-carbon units yields one NADH and two ATP.

Per glucose, glycolysis consumes 2 ATP (steps 1 and 3) and produces 4 ATP (two at step 7 and two at step 10), giving a net 2 ATP. It also yields 2 NADH at step 6. Both ATP-forming steps are substrate-level phosphorylation — a phosphate group is transferred from a high-energy intermediate (1,3-BPG or PEP) straight to ADP, with no electron transport chain involved.

Both enzymes phosphorylate glucose to glucose-6-phosphate, but they are built for different jobs. Hexokinase is a high-affinity enzyme present in all tissues, so it grabs glucose even when blood glucose is low. Glucokinase (hexokinase IV) is a low-affinity isoenzyme with a Km in the range of normal blood glucose, found in liver and pancreatic beta cells, so it works mainly after a meal.

Control sits at the three irreversible enzymes, through allosteric effectors, covalent modification and hormone-driven changes in enzyme synthesis. Glucose supply itself is also regulated through the GLUT transporters and glycogen breakdown.

Fructose-2,6-bisphosphate is the most powerful activator of PFK-1. It is made and destroyed by one bifunctional enzyme, PFK-2/FBPase-2. Insulin activates a phosphatase that dephosphorylates the enzyme, switching on its kinase activity: fructose-2,6-bisphosphate rises and glycolysis speeds up. Glucagon raises cAMP, protein kinase A phosphorylates the enzyme, the phosphatase activity takes over, fructose-2,6-bisphosphate falls, and gluconeogenesis prevails.

• Energy charge: high ATP and citrate signal plenty of energy and slow PFK-1; AMP signals shortage and speeds it.
• Pasteur effect: oxygen availability slows glycolysis, and falling oxygen accelerates it — most obvious in tissues with many mitochondria such as muscle and liver.
• Hormones: insulin increases glucokinase, PFK-1 and pyruvate kinase; when glucagon is high, the synthesis of these three enzymes falls.
• Glucose entry: GLUT1 (RBC, blood–brain barrier), GLUT2 (liver, beta cells, kidney, gut), GLUT3 (neurons), GLUT4 (muscle, fat, heart — insulin-responsive), GLUT5 (fructose).

In red cells, part of the 1,3-bisphosphoglycerate formed at step 6 is diverted away from phosphoglycerate kinase. Bisphosphoglycerate mutase converts it to 2,3-bisphosphoglycerate (2,3-BPG), which is then dephosphorylated back to 3-phosphoglycerate and rejoins glycolysis. Because this side-road bypasses the ATP-producing phosphoglycerate kinase step, the red cell trades ATP for 2,3-BPG.

2,3-BPG binds deoxyhaemoglobin in the central cleft between the beta chains and lowers haemoglobin's oxygen affinity, shifting the oxygen–haemoglobin dissociation curve to the right and helping oxygen unloading in tissues.

Pyruvate kinase deficiency is the classic glycolytic enzyme defect causing hereditary non-spherocytic haemolytic anaemia. It is autosomal recessive; the red-cell isoform (PKR) is encoded by the PKLR gene on chromosome 1q21. The pyruvate kinase step supplies about half of red-cell ATP, so without it the cell cannot run its Na+/K+ ATPases, loses potassium and water, becomes rigid and is removed by the spleen.

There are four pyruvate kinase isoforms: PKR only in red cells, PKL mainly in liver, PKM1 in muscle, heart and brain, and PKM2 in most other adult tissues, the embryo and cancers. PKR, PKL and PKM2 are all activated by fructose-1,6-bisphosphate.

Clinically, lactate — the end product of anaerobic glycolysis — is measured to judge tissue perfusion. Hyperlactataemia and lactic acidosis in sepsis, shock, severe anaemia or heart failure indicate that oxygen delivery is not meeting demand and are linked to higher mortality.

Every anomer is an epimer (a C-1 epimer), but not every epimer is an anomer: an anomer must differ at the anomeric carbon. In the cyclic form the α anomer has the same configuration at the anomeric carbon and at the stereocentre furthest from it; the β anomer has opposite configurations. Less than 0.01% of glucose is present in the open-chain aldehyde form at equilibrium.

• Rate-limiting enzyme of glycolysis → PFK-1; its strongest activator → fructose-2,6-bisphosphate.
• Substrate-level phosphorylation steps → phosphoglycerate kinase and pyruvate kinase.
• Only oxidative step → glyceraldehyde-3-phosphate dehydrogenase (NAD+ → NADH).
• Enzyme inhibited by fluoride → enolase.
• Glucose sensor of beta cells → glucokinase; mutation → MODY2.
• Hereditary non-spherocytic haemolytic anaemia with echinocytes → pyruvate kinase deficiency.
• Cells totally dependent on glycolysis → RBCs (no mitochondria), plus heavy reliance in lens, cornea and renal medulla.