Glycolysis and the TCA cycle
Energy metabolism: glycolysis, the citric acid cycle and their regulation; enzyme kinetics
Glucose is broken down in the cytosol by glycolysis, and its pyruvate is oxidised in the mitochondria by pyruvate dehydrogenase and the citric acid cycle; the liver can reverse much of this to make glucose when fasting. CPSP asks about rate-limiting enzymes, cofactors, regulators, glycogen storage diseases and Km and Vmax, often set in fetal hypoxia, fasting in pregnancy or hyperemesis.
Glycolysis (cytosol)
- Glucose to two pyruvate, net 2 ATP and 2 NADH per glucose, without oxygen.
- Three irreversible steps: hexokinase (glucokinase in liver and β cells), phosphofructokinase-1 (rate-limiting) and pyruvate kinase. The other seven steps (aldolase, GAPDH, enolase and others) are reversible and shared with gluconeogenesis.
- ATP used: hexokinase, PFK-1. ATP made by substrate-level phosphorylation: phosphoglycerate kinase and pyruvate kinase. GAPDH makes NADH, not ATP.
- Glucose-6-phosphate is the branch point to glycolysis, glycogen (via glucose-1-phosphate) and the pentose phosphate pathway (NADPH via G6PD), as in lactating breast making milk fat.
- Without oxygen, lactate dehydrogenase turns pyruvate into lactate to regenerate NAD+ so GAPDH can keep running. This is how the hypoxic fetus or ischaemic myocardium keeps making ATP; the acidosis is from hydrogen ions released with lactate.
- In red cells (no mitochondria) a side branch from 1,3-bisphosphoglycerate makes 2,3-BPG (bisphosphoglycerate mutase), which shifts the oxygen curve right; it rises in pregnancy.
- Fluoride in grey-top tubes inhibits enolase, stopping cells consuming glucose in a waiting sample.
Regulation of PFK-1 and pyruvate kinase
| Regulator | Effect | Setting |
|---|---|---|
| AMP, ADP | Activate PFK-1 | Energy shortage, e.g. ischaemic myometrium in obstructed labour |
| ATP, citrate | Inhibit PFK-1 | Fat oxidation in late pregnancy spares glucose |
| Fructose-2,6-bisphosphate | Activates PFK-1, inhibits fructose-1,6-bisphosphatase | Raised by insulin, lowered by glucagon |
| Glucagon (cAMP, PKA) | Phosphorylates and inactivates liver pyruvate kinase | Fasting: PEP goes on to glucose |
| Fructose-1,6-bisphosphate | Activates pyruvate kinase (feed-forward) | Fed state |
Severe acidosis eventually inhibits PFK-1.
Pyruvate dehydrogenase and the citric acid cycle (mitochondrial matrix)
- Pyruvate must enter the mitochondria (pyruvate carrier) because PDH and the TCA cycle are in the matrix. Blocked entry raises lactate.
- PDH turns pyruvate into acetyl-CoA, irreversibly, so even-chain fat cannot give net glucose. Cofactors: thiamine (B1), riboflavin, niacin, pantothenate and lipoic acid.
- PDH is switched off by phosphorylation by PDH kinase (activated by acetyl-CoA and NADH from fat oxidation) and switched on by a phosphatase (insulin, calcium).
- Each turn of the cycle gives 3 NADH, 1 FADH2 and 1 GTP (GTP from succinyl-CoA to succinate).
- Isocitrate dehydrogenase is the main rate-limiting step, activated by ADP and calcium (as in contracting myometrium), inhibited by ATP and NADH.
- α-Ketoglutarate dehydrogenase also needs thiamine. In hyperemesis, thiamine depletion stops it and PDH in neurons, causing Wernicke encephalopathy, which glucose without thiamine worsens.
- Succinate dehydrogenase is the only TCA enzyme in the inner membrane and is complex II; its mutations cause paragangliomas. Fumarate hydratase mutations cause skin and uterine leiomyomas with renal cancer.
- Ammonia drains α-ketoglutarate (to glutamate and glutamine), slowing the cycle in brain.
Gluconeogenesis and glycogen
- Liver (and kidney) make glucose from lactate, glycerol (glycerol kinase, liver only), alanine and propionate; not from acetyl-CoA or leucine and lysine. Alanine carries carbon and nitrogen from muscle to liver.
- Bypass enzymes: pyruvate carboxylase (biotin, activated by acetyl-CoA), PEPCK, fructose-1,6-bisphosphatase (rate-limiting) and glucose-6-phosphatase (absent in muscle, so muscle glycogen cannot supply blood glucose).
- Overnight fast: hepatic glycogenolysis keeps glucose normal for 12–24 hours; gluconeogenesis then takes over. Alcohol excess makes NADH, diverting pyruvate and oxaloacetate and blocking gluconeogenesis; liver failure loses both.
- Glycogen synthase (α-1,4, rate-limiting) plus branching enzyme (α-1,6) build glycogen; phosphorylase (pyridoxal phosphate) plus debranching enzyme break it. Glucagon and adrenaline (cAMP, PKA) phosphorylate: phosphorylase on, synthase off; insulin dephosphorylates.
- Preterm and growth-restricted babies have low hepatic glycogen (laid down in the third trimester) and become hypoglycaemic.
| Glycogen storage disease | Enzyme | Key features |
|---|---|---|
| I von Gierke | Glucose-6-phosphatase | Fasting hypoglycaemia, big liver, lactic acidosis, high urate |
| II Pompe | Lysosomal acid maltase | Cardiomegaly |
| III Cori | Debranching enzyme | Milder type I picture |
| V McArdle | Muscle phosphorylase | Exercise cramps, myoglobinuria, no lactate rise, second wind |
Enzyme kinetics
- Km is the substrate concentration at half Vmax; low Km means high affinity. Vmax depends on the amount of enzyme, so enzyme induction (lamotrigine glucuronidation in pregnancy) raises Vmax without changing Km.
- Hexokinase (brain, placenta) has a Km of about 0.1 mmol/L and stays saturated in hypoglycaemia; glucokinase (liver) has a Km of about 8 mmol/L (145 mg/dL) and works only when glucose is high.
- Carrier-mediated transport (facilitated diffusion) is saturable and so shows a Vmax.
- Allosteric enzymes such as PFK-1 give an S-shaped curve; activators shift it left, inhibitors right.
| Inhibitor | Km | Vmax | Lineweaver–Burk |
|---|---|---|---|
| Competitive (overcome by more substrate) | Rises | Unchanged | Lines meet on the y-axis |
| Non-competitive | Unchanged | Falls | Lines meet on the x-axis |
| Uncompetitive (binds ES complex only) | Falls | Falls | Parallel lines |
On the plot, the y-intercept is 1/Vmax and the x-intercept is −1/Km.
Remember Rate-limiting enzymes: PFK-1 for glycolysis, fructose-1,6-bisphosphatase for gluconeogenesis, isocitrate dehydrogenase for the TCA cycle, glycogen synthase and phosphorylase for glycogen.
Exam trap Muscle cannot release glucose from glycogen because it lacks glucose-6-phosphatase, not because it lacks phosphorylase.
Exam trap Low Km means high affinity, not high speed; competitive inhibition raises Km and leaves Vmax unchanged.
Test yourself on this topic
5 of the 47 questions in our bank on this topic. Choose an answer to see why every option is right or wrong.
After an overnight fast, a woman at 32 weeks has high free fatty acids and ketones, and her muscles burn fat while sparing glucose for the fetus. How is pyruvate dehydrogenase switched off in her muscles?
Answer: Phosphorylation by PDH kinase
Fatty acid oxidation raises mitochondrial acetyl-CoA and NADH. These activate pyruvate dehydrogenase kinase, which phosphorylates and inactivates the PDH complex, and also inhibit PDH directly as products. Pyruvate is then not oxidised but returned as lactate and alanine for hepatic gluconeogenesis, sparing glucose. In late pregnancy this 'accelerated starvation' helps preserve glucose for the fetus.
PDH in fasting
- Fat oxidation: acetyl-CoA, NADH rise
- Activate PDH kinase
- PDH phosphorylated, inactive
- Glucose spared for the fetus
Remember Burning fat switches off glucose oxidation.
Exam trap Insulin activates PDH through its phosphatase; it does not inhibit it.
Why the other options are wrong
- Dephosphorylation by insulin Insulin activates PDH by stimulating its phosphatase.
- Direct binding of glucagon Muscle lacks glucagon receptors.
- Removal of its thiamine cofactor Thiamine stays bound as a cofactor.
- Competition by lactate at its site Lactate does not inhibit PDH this way.
In late pregnancy, maternal red cells contain more 2,3-bisphosphoglycerate, helping them release oxygen to the placenta. Red cells have no mitochondria. How is 2,3-BPG produced within them?
Answer: Side branch of glycolysis from 1,3-BPG
In red cells, part of the glycolytic intermediate 1,3-bisphosphoglycerate is diverted by bisphosphoglycerate mutase to 2,3-BPG (the Rapoport–Luebering shunt), which is then dephosphorylated to 3-phosphoglycerate to rejoin glycolysis. This bypasses the ATP-generating phosphoglycerate kinase step, trading ATP for 2,3-BPG. 2,3-BPG binds deoxyhaemoglobin, shifting the curve right, while fetal haemoglobin binds it poorly.
Rapoport–Luebering shunt
- From 1,3-bisphosphoglycerate
- Enzyme: bisphosphoglycerate mutase
- Bypasses an ATP-making step
- 2,3-BPG shifts the curve right
Remember Red cells trade ATP for 2,3-BPG.
Exam trap Pyruvate kinase deficiency raises 2,3-BPG and improves oxygen delivery despite the anaemia.
Why the other options are wrong
- Pentose phosphate pathway from ribose The pentose phosphate pathway makes NADPH, not 2,3-BPG.
- TCA cycle from succinyl-CoA Red cells have no TCA cycle.
- Breakdown of haem by haem oxygenase Haem breakdown gives biliverdin and carbon monoxide.
- Phosphorylation of glycerol by glycerol kinase Red cells do not make 2,3-BPG from glycerol.
A woman with severe hyperemesis gravidarum at 14 weeks becomes confused and ataxic, with nystagmus, after intravenous dextrose without vitamins. Failure of which thiamine-dependent TCA cycle enzyme in neurons most directly reduces their ATP supply?
Answer: Alpha-ketoglutarate dehydrogenase
Weeks of vomiting deplete thiamine. Thiamine pyrophosphate is a cofactor for pyruvate dehydrogenase, transketolase and the alpha-ketoglutarate dehydrogenase complex, which is the TCA cycle enzyme that needs it. A glucose load increases flux through these enzymes, so neurons with high oxidative demand in the mammillary bodies and periaqueductal region run short of ATP, causing Wernicke encephalopathy. Thiamine must be given before or with dextrose.
Wernicke in hyperemesis
- Vomiting depletes thiamine
- TPP needed by alpha-KG dehydrogenase
- Also PDH and transketolase
- Give thiamine before glucose
Remember Hyperemesis plus dextrose: thiamine first.
Exam trap Confusion, ataxia and eye signs rarely all appear together; one or two is enough to treat.
Why the other options are wrong
- Mitochondrial citrate synthase Citrate synthase does not need thiamine.
- Isocitrate dehydrogenase Isocitrate dehydrogenase needs NAD+, not thiamine.
- Succinate dehydrogenase Succinate dehydrogenase uses FAD.
- Malate dehydrogenase (NAD+) Malate dehydrogenase uses NAD+.
In late pregnancy, high free fatty acids from placental lactogen-driven lipolysis are oxidised by maternal muscle, and its glucose use falls. Which molecule, exported from mitochondria to the cytosol, directly inhibits phosphofructokinase-1 in this setting?
Answer: Citrate
Fatty acid oxidation produces acetyl-CoA, which combines with oxaloacetate to form citrate. Citrate moves into the cytosol and allosterically inhibits PFK-1, slowing glycolysis, while acetyl-CoA and NADH inhibit pyruvate dehydrogenase. This glucose–fatty acid (Randle) cycle reduces muscle glucose uptake and use, contributing to the insulin resistance of late pregnancy that diverts glucose to the fetus.
Randle cycle
- Fat oxidation: citrate rises
- Citrate inhibits PFK-1
- Acetyl-CoA inhibits PDH
- Less glucose use by muscle
Remember Burning fat slows glycolysis through citrate.
Exam trap Malonyl-CoA works the other way, stopping fat oxidation when glucose is plentiful.
Why the other options are wrong
- AMP AMP activates PFK-1.
- Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate activates PFK-1.
- Lactate Lactate does not directly inhibit PFK-1.
- Malonyl-CoA Malonyl-CoA inhibits carnitine palmitoyltransferase I, not PFK-1.
In a child with a rare defect of the mitochondrial pyruvate carrier, blood lactate and pyruvate are high and energy production is poor, although all glycolytic and TCA enzymes are normal. Why must pyruvate from glycolysis enter the mitochondria to be oxidised?
Answer: PDH and the TCA cycle are in the matrix
Glycolysis takes place in the cytosol, but the pyruvate dehydrogenase complex, the TCA cycle enzymes (except succinate dehydrogenase, on the inner membrane) and oxidative phosphorylation are in the mitochondria. Pyruvate is carried across the inner mitochondrial membrane by the mitochondrial pyruvate carrier. If it cannot enter, it accumulates and is converted to lactate in the cytosol, and much less ATP is made.
Where pyruvate is oxidised
- Glycolysis in the cytosol
- PDH and TCA in the matrix
- Entry by mitochondrial pyruvate carrier
- Blocked entry: lactate rises
Remember Pyruvate must go into the mitochondrion to burn.
Exam trap Succinate dehydrogenase is the only TCA enzyme bound to the inner membrane.
Why the other options are wrong
- Pyruvate cannot cross the plasma membrane Pyruvate can cross the plasma membrane via monocarboxylate transporters.
- Glycolysis itself occurs in the mitochondria Glycolysis is cytosolic.
- Lactate dehydrogenase is mitochondrial Lactate dehydrogenase is cytosolic.
- Pyruvate is toxic in the cytosol Pyruvate is not intrinsically toxic in the cytosol.
Where this sits in the CPSP syllabus
- Obstetrics & Gynaecology, Paper 2: Systems physiology for obstetrics, Energy metabolism: glycolysis, citric acid cycle and regulation; enzyme kinetics
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