Biochemistry chapters
Glycolysis, Krebs cycle and the electron transport chain
How cells extract energy from glucose: glycolysis and its control, pyruvate dehydrogenase, the Krebs (TCA) cycle, the electron transport chain and oxidative phosphorylation with their inhibitors and uncouplers, then gluconeogenesis, glycogen metabolism, the pentose phosphate pathway and the key enzyme defects, with ATP yields worked out by hand.
- Glycolysis (cytosol, all cells) turns glucose into 2 pyruvate, giving a net 2 ATP and 2 NADH; key irreversible enzymes are hexokinase/glucokinase, PFK-1 (rate-limiting; activated by AMP and fructose 2,6-bisphosphate, inhibited by ATP and citrate) and pyruvate kinase; without oxygen (and always in red cells, which lack mitochondria) pyruvate becomes lactate.
- Pyruvate dehydrogenase (needs TPP, lipoate, CoA, FAD, NAD: inhibited by arsenic) makes acetyl-CoA for the TCA cycle (mitochondria; rate-limiting isocitrate dehydrogenase), whose NADH and FADH₂ feed the electron transport chain; cyanide and carbon monoxide block complex IV, rotenone complex I, antimycin A complex III, oligomycin ATP synthase, while uncouplers (2,4-dinitrophenol, aspirin overdose, thermogenin in brown fat) make heat instead of ATP; complete oxidation of glucose yields about 30–32 ATP.
- Gluconeogenesis (liver, kidney) bypasses the irreversible steps with pyruvate carboxylase, PEPCK, fructose 1,6-bisphosphatase and glucose 6-phosphatase; the pentose phosphate pathway (rate-limiting G6PD) makes NADPH (protects red cells: G6PD deficiency → haemolysis) and ribose; von Gierke disease (glucose 6-phosphatase) causes severe fasting hypoglycaemia with lactic acidosis.
On this page
Glucose entry
| Transporter | Site | Features |
|---|---|---|
| GLUT1 | Red cells, brain, placenta | Basal uptake, insulin-independent |
| GLUT2 | Liver, pancreatic β cells, kidney, intestine (basolateral) | High Km; glucose sensing; two-way |
| GLUT3 | Neurons | High affinity |
| GLUT4 | Skeletal muscle, heart, adipose tissue | Insulin-dependent (moves to the membrane with insulin) |
| GLUT5 | Intestine, sperm | Fructose |
| SGLT1, SGLT2 | Intestine, kidney tubule | Sodium-glucose co-transport (secondary active) |
Glycolysis
Location: cytosol of all cells. Glucose → 2 pyruvate.
Key (irreversible, regulated) steps
| Enzyme | Reaction | Regulation |
|---|---|---|
| Hexokinase (most tissues) / glucokinase (liver, β cells) | Glucose → glucose 6-phosphate (uses ATP) | Hexokinase: low Km, high affinity, inhibited by glucose 6-phosphate. Glucokinase: high Km, low affinity, high capacity; induced by insulin; acts after meals |
| Phosphofructokinase-1 (PFK-1) | Fructose 6-P → fructose 1,6-bisphosphate (uses ATP) | Rate-limiting. ↑ by AMP, fructose 2,6-bisphosphate (insulin); ↓ by ATP, citrate, H⁺ |
| Pyruvate kinase | Phosphoenolpyruvate → pyruvate (makes ATP) | ↑ by fructose 1,6-bisphosphate (feed-forward), insulin; ↓ by ATP, alanine, glucagon (phosphorylation in liver) |
ATP is made by substrate-level phosphorylation at phosphoglycerate kinase and pyruvate kinase.
Investment: hexokinase −1 ATP, PFK-1 −1 ATP = −2 ATP
Payoff (×2, since glucose splits into two 3-carbon units):
phosphoglycerate kinase +1 × 2 = +2
pyruvate kinase +1 × 2 = +2
Total made = 4 ATP
Net = 4 − 2 = 2 ATP per glucose (+ 2 NADH)
Anaerobic glycolysis: the 2 NADH are used to make lactate → net 2 ATP only
From glycogen (glucose 1-P, no hexokinase step): net 3 ATP
Fate of pyruvate and NADH
- Aerobic: pyruvate enters mitochondria → acetyl-CoA (pyruvate dehydrogenase).
- Anaerobic: lactate dehydrogenase turns pyruvate into lactate, regenerating NAD⁺ so glycolysis can continue. Happens in red cells (no mitochondria), exercising muscle, lens, cornea, renal medulla, testis, and in hypoxia (shock: lactic acidosis).
- Cytosolic NADH enters mitochondria by shuttles: malate-aspartate (heart, liver: about 2.5 ATP) or glycerol-phosphate (muscle, brain: about 1.5 ATP).
Red cell glycolysis
- Red cells rely entirely on glycolysis.
- The Rapoport-Luebering shunt makes 2,3-bisphosphoglycerate, which lowers haemoglobin's oxygen affinity (shifts the curve right), helping oxygen release to tissues.
- Pyruvate kinase deficiency: the commonest glycolytic enzyme defect; chronic haemolytic anaemia (low ATP, rigid cells); 2,3-BPG rises.
Inhibitors
Fluoride inhibits enolase (used in blood glucose tubes to stop glycolysis); arsenate uncouples glyceraldehyde-3-P dehydrogenase from ATP production; iodoacetate inhibits glyceraldehyde-3-P dehydrogenase.
Pyruvate dehydrogenase (PDH) complex
Pyruvate → acetyl-CoA + CO₂ + NADH, in the mitochondria; irreversible (so acetyl-CoA cannot be turned back into glucose).
Cofactors: thiamine pyrophosphate (B1), lipoic acid, coenzyme A (B5), FAD (B2), NAD⁺ (B3): "Tender Loving Care For Nancy". The same cofactors serve α-ketoglutarate dehydrogenase.
- Activated by insulin, ADP, pyruvate, calcium (dephosphorylation); inhibited by acetyl-CoA, NADH, ATP (phosphorylation).
- Arsenic binds lipoic acid → blocks PDH (vomiting, rice-water stools, garlic breath).
- Thiamine deficiency blocks PDH → lactic acidosis (Wernicke, beriberi).
- PDH deficiency: congenital lactic acidosis, neurological damage; treated with a ketogenic diet (fat and the ketogenic amino acids leucine and lysine).
TCA (Krebs, citric acid) cycle
Location: mitochondrial matrix. The final common pathway for oxidising carbohydrate, fat and protein.
Sequence: acetyl-CoA + oxaloacetate → citrate (citrate synthase) → isocitrate → α-ketoglutarate (isocitrate dehydrogenase: rate-limiting; makes NADH, CO₂) → succinyl-CoA (α-ketoglutarate dehydrogenase; NADH, CO₂) → succinate (makes GTP) → fumarate (succinate dehydrogenase: FADH₂; also complex II of the ETC; inhibited by malonate) → malate → oxaloacetate (NADH).
"Citrate Is Krebs' Starting Substrate For Making Oxaloacetate": Citrate, Isocitrate, α-Ketoglutarate, Succinyl-CoA, Succinate, Fumarate, Malate, Oxaloacetate.
Per acetyl-CoA: 3 NADH, 1 FADH₂, 1 GTP, 2 CO₂ → about 10 ATP.
- Needs oxygen indirectly (NAD⁺ and FAD regeneration by the ETC).
- Amphibolic: intermediates are used for synthesis (citrate → fatty acids; succinyl-CoA → haem; α-ketoglutarate and oxaloacetate → amino acids; oxaloacetate → glucose).
- Fluoroacetate (rat poison) forms fluorocitrate, which blocks aconitase.
Electron transport chain and oxidative phosphorylation
Location: inner mitochondrial membrane. NADH and FADH₂ pass electrons down a chain to oxygen (forming water); the energy pumps protons out, and ATP synthase (complex V) uses the proton gradient to make ATP (chemiosmotic theory, Mitchell).
| Complex | Name | Inhibitors |
|---|---|---|
| I | NADH dehydrogenase | Rotenone, amobarbital, metformin (weakly) |
| II | Succinate dehydrogenase (FADH₂ enters; no proton pumping) | Malonate |
| Coenzyme Q (ubiquinone) | Mobile carrier | (none commonly tested) |
| III | Cytochrome b-c1 | Antimycin A |
| Cytochrome c | Mobile carrier | (none commonly tested) |
| IV | Cytochrome oxidase (a-a3; contains copper and haem) | Cyanide, carbon monoxide, azide, hydrogen sulphide |
| V | ATP synthase | Oligomycin |
P/O ratios (modern values): NADH → about 2.5 ATP; FADH₂ → about 1.5 ATP (older books: 3 and 2).
Uncouplers make the inner membrane leaky to protons, so electron transport continues (oxygen used) but no ATP is made; energy is released as heat:
- 2,4-dinitrophenol (once used for weight loss: fatal hyperthermia).
- Aspirin overdose (hyperthermia).
- Thermogenin (UCP1) in brown fat: physiological heat production in newborns.
Inhibitors stop both electron flow and ATP synthesis. Uncouplers stop ATP synthesis but increase oxygen use and heat. Cyanide blocks complex IV (cytochrome oxidase): cells cannot use oxygen (venous blood stays bright red, lactic acidosis); treat with hydroxocobalamin or nitrites + thiosulphate.
Glycolysis: 2 ATP + 2 NADH
2 NADH × 2.5 (malate-aspartate shuttle) = 5 ATP (or × 1.5 = 3 with glycerol-phosphate shuttle)
Pyruvate dehydrogenase (×2): 2 NADH × 2.5 = 5 ATP
TCA cycle (×2 acetyl-CoA):
6 NADH × 2.5 = 15
2 FADH₂ × 1.5 = 3
2 GTP = 2
TCA total = 20
Grand total = 2 + 5 + 5 + 20 = 32 ATP (30 with the glycerol-phosphate shuttle)
(Older textbooks using 3 and 2: 36–38 ATP)
Gluconeogenesis
Making glucose from non-carbohydrate sources (lactate, glycerol, glucogenic amino acids such as alanine, propionate) during fasting. Mainly in the liver (about 90%) and kidney (more in prolonged starvation). Fatty acids (even-chain) cannot be made into glucose (PDH is irreversible), but propionyl-CoA from odd-chain fatty acids can.
Bypasses of the irreversible glycolytic steps:
- Pyruvate carboxylase (mitochondria; needs biotin; activated by acetyl-CoA): pyruvate → oxaloacetate.
- PEP carboxykinase (PEPCK): oxaloacetate → phosphoenolpyruvate (uses GTP).
- Fructose 1,6-bisphosphatase: the rate-limiting step; inhibited by fructose 2,6-bisphosphate and AMP.
- Glucose 6-phosphatase (ER of liver and kidney, not muscle): releases free glucose into the blood.
Cost: 6 high-energy bonds (4 ATP + 2 GTP) per glucose.
Cori cycle: lactate from muscle and red cells → liver → glucose → back to tissues. Glucose-alanine cycle: alanine from muscle → liver → glucose (also carries nitrogen).
Alcohol inhibits gluconeogenesis (high NADH/NAD⁺ ratio diverts pyruvate to lactate and oxaloacetate to malate) → hypoglycaemia in fasting drinkers.
Glycogen metabolism
Glycogen is stored in liver (maintains blood glucose between meals) and muscle (fuel for the muscle itself; muscle lacks glucose 6-phosphatase).
- Synthesis: glucose 6-P → glucose 1-P → UDP-glucose → glycogen synthase (rate-limiting; α-1,4 links; activated by insulin) + branching enzyme (α-1,6 links).
- Breakdown: glycogen phosphorylase (rate-limiting; needs PLP; activated by glucagon (liver) and adrenaline, AMP and calcium (muscle)) releases glucose 1-P from α-1,4 links; debranching enzyme handles branches.
| Glycogen storage disease | Enzyme | Key features |
|---|---|---|
| Type I (von Gierke) | Glucose 6-phosphatase | Severe fasting hypoglycaemia, lactic acidosis, hepatomegaly, hyperlipidaemia, hyperuricaemia, doll face; frequent feeds, cornstarch |
| Type II (Pompe) | Lysosomal acid α-glucosidase (acid maltase) | Cardiomegaly, hypotonia, early death in infantile form; enzyme replacement |
| Type III (Cori) | Debranching enzyme | Milder type I; normal lactate |
| Type V (McArdle) | Muscle glycogen phosphorylase | Exercise-induced cramps, myoglobinuria, no rise in lactate after exercise; "second wind" phenomenon |
Pentose phosphate pathway (hexose monophosphate shunt)
Location: cytosol; active in liver, adipose tissue, adrenal cortex, gonads, red cells, lactating breast.
- Oxidative phase (irreversible): glucose 6-phosphate dehydrogenase (G6PD) (rate-limiting) → NADPH + ribulose 5-phosphate.
- Non-oxidative phase (reversible): transketolase (needs TPP) and transaldolase make ribose 5-phosphate (for nucleotides) and recycle sugars.
- No ATP is made or used.
NADPH is needed for: fatty acid and cholesterol synthesis, keeping glutathione reduced (protects red cells from oxidants), respiratory burst in phagocytes (NADPH oxidase), cytochrome P450 reactions, nitric oxide synthesis.
G6PD deficiency (X-linked; common in Pakistan and other malaria regions): red cells cannot make NADPH → glutathione cannot be kept reduced → oxidant stress (drugs such as primaquine, dapsone, sulphonamides, nitrofurantoin; fava beans; infections) → haemolysis with Heinz bodies and bite cells; neonatal jaundice. Gives some protection against malaria.
Other sugar disorders
| Disorder | Enzyme | Features |
|---|---|---|
| Classic galactosaemia | Galactose-1-phosphate uridyltransferase | After milk feeds: vomiting, jaundice, hepatomegaly, failure to thrive, cataracts, E. coli sepsis, intellectual disability; remove galactose and lactose from the diet |
| Galactokinase deficiency | Galactokinase | Milder: cataracts only |
| Hereditary fructose intolerance | Aldolase B | Symptoms when fructose or sucrose introduced (weaning, fruit): hypoglycaemia, vomiting, jaundice, liver failure; avoid fructose and sucrose |
| Essential fructosuria | Fructokinase | Harmless; fructose in urine |
| Lactose intolerance | Lactase deficiency (often acquired in adulthood; secondary after gastroenteritis) | Bloating, flatulence, osmotic diarrhoea after milk |
Cataracts in galactosaemia and diabetes come from aldose reductase making sorbitol/galactitol, which draws water into the lens.
Expect: GLUT4 and insulin, hexokinase vs glucokinase, PFK-1 regulation, net ATP of glycolysis (aerobic, anaerobic, from glycogen), red cell glycolysis and 2,3-BPG, PDH cofactors and arsenic, TCA cycle rate-limiting step and products, ETC complexes and inhibitors (cyanide, CO, rotenone, antimycin, oligomycin), uncouplers and brown fat, total ATP per glucose, gluconeogenic bypass enzymes, why fatty acids cannot make glucose, Cori cycle, glycogen enzymes and storage diseases (von Gierke, Pompe, McArdle), the pentose phosphate pathway and G6PD deficiency, galactosaemia, fructose intolerance.
Quick revision
- Glycolysis: cytosol; net 2 ATP + 2 NADH; PFK-1 rate-limiting; red cells use only glycolysis.
- PDH: TPP, lipoate, CoA, FAD, NAD; arsenic blocks lipoate.
- TCA: mitochondria; isocitrate dehydrogenase rate-limiting; 3 NADH, 1 FADH₂, 1 GTP per acetyl-CoA.
- ETC: I rotenone, III antimycin A, IV cyanide/CO, V oligomycin; uncouplers make heat.
- Glucose → about 30–32 ATP.
- Gluconeogenesis: pyruvate carboxylase, PEPCK, F-1,6-bisphosphatase (rate-limiting), glucose 6-phosphatase.
- Von Gierke: glucose 6-phosphatase. Pompe: acid maltase (heart). McArdle: muscle phosphorylase.
- HMP shunt: G6PD → NADPH; deficiency → haemolysis with oxidant drugs.
Written by The House Job from standard biochemistry references (Lippincott Illustrated Reviews: Biochemistry).
FAQ
What is the net ATP gain from glycolysis?
Two ATP per glucose (plus two NADH); three if the glucose comes from glycogen.
Which enzyme is rate-limiting in glycolysis?
Phosphofructokinase-1.
How does cyanide poison cells?
It blocks cytochrome oxidase (complex IV), so cells cannot use oxygen to make ATP, leading to lactic acidosis.
Why does G6PD deficiency cause haemolysis?
Red cells need NADPH from the pentose phosphate pathway to keep glutathione reduced; without it, oxidant drugs or infections damage haemoglobin and membranes, and the cells break down.