What is the role of leghaemoglobin in the root nodules of legumes?

It binds O₂ tightly, keeping free O₂ low while supplying bacteroid respiration

It transfers electrons from ferredoxin to the Fe protein of nitrogenase

It forms the iron–molybdenum cofactor at the active site of nitrogenase

It binds O₂ tightly, keeping free O₂ low while supplying bacteroid respiration

It stores the fixed ammonia as a haem complex until the host plant needs it

When ammonium is scarce, bacteria and plants assimilate it mainly through glutamine synthetase and glutamate synthase (GOGAT) rather than through glutamate dehydrogenase. Why?

Glutamine synthetase has a much higher affinity for NH₄⁺, and its ATP hydrolysis drives assimilation at low concentration

Glutamate dehydrogenase works only in the deamination direction and cannot add ammonia to α-ketoglutarate

Glutamine synthetase has a much higher affinity for NH₄⁺, and its ATP hydrolysis drives assimilation at low concentration

Glutamate synthase fixes atmospheric N₂ directly into glutamate without any need for nitrogenase

Glutamine synthetase uses NADPH, which is far more plentiful than ATP when ammonium is limiting

All aminotransferases require pyridoxal phosphate. What does the cofactor do in the reaction?

It forms a Schiff base with the amino acid and holds the amino group as pyridoxamine phosphate

It accepts the amino group as free NH₄⁺, which is then handed directly to carbamoyl phosphate synthetase

It carries a one-carbon unit from serine to the α-keto acid acceptor of the reaction

It oxidises the amino acid with NAD⁺ to an imine, which water then hydrolyses to the keto acid

It forms a Schiff base with the amino acid and holds the amino group as pyridoxamine phosphate

Two newborns have hyperammonaemia with low plasma citrulline. One excretes large amounts of orotic acid in urine; the other does not. Which enzyme is most likely deficient in the infant with orotic aciduria?

Ornithine transcarbamoylase, because unused carbamoyl phosphate leaks to the cytosol and enters pyrimidine synthesis

Carbamoyl phosphate synthetase I, because the lack of carbamoyl phosphate overstimulates cytosolic pyrimidine synthesis

Argininosuccinate synthetase, because citrulline that cannot be used is converted into orotic acid by the liver

Arginase, because ornithine runs short and carbamoyl phosphate is turned into orotate inside the mitochondrion

Which of the following statements about the urea cycle are correct?

N-Acetylglutamate is an essential allosteric activator of carbamoyl phosphate synthetase I

Argininosuccinate synthetase cleaves ATP to AMP and pyrophosphate

N-Acetylglutamate is an essential allosteric activator of carbamoyl phosphate synthetase I

Both nitrogen atoms of urea come from free NH₄⁺ that enters through carbamoyl phosphate

Argininosuccinate synthetase cleaves ATP to AMP and pyrophosphate

Ornithine transcarbamoylase and arginase both act in the mitochondrial matrix of hepatocytes

Methotrexate is used against rapidly dividing cancer cells. Its main action is to

inhibit dihydrofolate reductase, so tetrahydrofolate is not regenerated after thymidylate synthesis

inactivate thymidylate synthase covalently after its conversion to a fluorinated deoxynucleotide

inhibit ribonucleotide reductase by scavenging the tyrosyl radical that its catalysis requires

inhibit xanthine oxidase, so that purine bases cannot be degraded and accumulate inside cells

A boy with self-injurious behaviour and gouty arthritis has a raised serum uric acid. His cultured cells contain high PRPP and show an increased rate of de novo purine synthesis. Which defect best accounts for all these findings?

Deficiency of hypoxanthine-guanine phosphoribosyltransferase, so salvage no longer consumes PRPP

Loss of xanthine oxidase activity, so that hypoxanthine accumulates and drives de novo synthesis

Deficiency of adenosine deaminase, so that deoxyadenosine nucleotides accumulate in his lymphocytes

Deficiency of hypoxanthine-guanine phosphoribosyltransferase, so salvage no longer consumes PRPP

A PRPP synthetase that has lost activity, so purine nucleotides cannot be made by either route

Which electron carrier transfers electrons from the cytochrome b₆f complex to photosystem I?

Plastocyanin, a copper protein in the thylakoid lumen

Plastoquinone, the mobile carrier that diffuses within the membrane

Ferredoxin, the soluble iron–sulfur protein of the stroma

Pheophytin, the first electron acceptor within photosystem II

Plastocyanin, a copper protein in the thylakoid lumen

Illuminated chloroplasts supplied with ADP, Pi and NADP⁺ are treated with DCMU. Which observation is expected?

O₂ evolution and NADP⁺ reduction stop, but cyclic photophosphorylation can still make ATP

O₂ evolution continues, but NADP⁺ reduction stops because DCMU blocks ferredoxin–NADP⁺ reductase

O₂ evolution and NADP⁺ reduction stop, but cyclic photophosphorylation can still make ATP

All ATP synthesis stops at once, because DCMU makes the thylakoid membrane leaky to protons

NADP⁺ reduction continues with electrons that photosystem I removes from water by itself

Which of the following statements about the Calvin cycle and its regulation are correct?

Illumination raises stromal pH and Mg²⁺, both of which activate Rubisco and fructose-1,6-bisphosphatase

The CO₂ that carbamylates the active-site lysine of Rubisco is the same molecule that is fixed into 3-phosphoglycerate

The oxygenase reaction of Rubisco gives two molecules of 3-phosphoglycerate for each O₂ consumed

An infant thrives on breast milk but develops vomiting, hypoglycaemia and an enlarged liver soon after fruit juice and sucrose are added to the diet. Which enzyme is most likely deficient?

Aldolase B, which cleaves fructose-1-phosphate into two three-carbon products

Fructokinase, which phosphorylates fructose to fructose-1-phosphate in the liver

Galactose-1-phosphate uridylyltransferase, which converts galactose to a glucose derivative

Aldolase B, which cleaves fructose-1-phosphate into two three-carbon products

Glucose-6-phosphatase, which releases free glucose from the liver between meals

Which of the following statements about inherited disorders of amino acid and lipid metabolism are correct?

In classic phenylketonuria, tyrosine becomes a dietary essential amino acid

In alkaptonuria, homogentisate oxidase is deficient and the urine darkens on standing in air

In Tay–Sachs disease, hexosaminidase A is deficient and GM2 ganglioside accumulates in neurons

In classic phenylketonuria, tyrosine becomes a dietary essential amino acid

In alkaptonuria, homogentisate oxidase is deficient and the urine darkens on standing in air

Maple syrup urine disease is a defect in the breakdown of the aromatic amino acids

In Tay–Sachs disease, hexosaminidase A is deficient and GM2 ganglioside accumulates in neurons

Patient X has fasting hypoglycaemia, an enlarged liver, lactic acidosis and no rise in blood glucose after glucagon. Patient Y has muscle cramps during exercise, and his blood lactate does not rise during an ischaemic forearm exercise test. Which deficiencies explain X and Y respectively?

Glucose-6-phosphatase in X; muscle glycogen phosphorylase in Y

Muscle glycogen phosphorylase in X; glucose-6-phosphatase in Y

Glucose-6-phosphatase in X; lysosomal acid α-glucosidase in Y

Lysosomal acid α-glucosidase in X; liver glycogen phosphorylase in Y

Glucose-6-phosphatase in X; muscle glycogen phosphorylase in Y

Nitrogen Metabolism, Photosynthesis and the Calvin Cycle, and Metabolic Disorders

This chapter completes Section XL1.3 of XL1 Biochemistry, “Metabolic Pathways and Associated Disorders”, by taking its remaining clauses: “Metabolism of nitrogen-containing compounds: nitrogen fixation, amino acids, and nucleotides. Photosynthesis, photophosphorylation, Calvin cycle. Metabolic disorders (Sugars, amino acids, and lipids).” It follows nitrogen from N₂ to ammonia, amino acids, urea and nucleotides; then light energy from water to NADPH and ATP and into the Calvin cycle; and it ends with the inborn errors that reveal each pathway by its absence. The numericals are the ATP cost of nitrogen fixation and of urea synthesis, the origin of purine ring atoms, photon budgets and the ATP and NADPH cost of fixing CO₂.

1. Nitrogen fixation and ammonia assimilation

Only prokaryotes fix N₂: free-living bacteria such as Azotobacter and Clostridium, cyanobacteria, and rhizobia in legume root nodules. The nitrogenase complex has two proteins. The Fe protein (dinitrogenase reductase) takes electrons from reduced ferredoxin or flavodoxin and, hydrolysing 2 ATP per electron, passes them one at a time to the MoFe protein (dinitrogenase), whose iron–molybdenum cofactor binds and reduces N₂. The accepted stoichiometry, with obligatory H₂ formation, is N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 Pi.

Nitrogenase is irreversibly inactivated by O₂, so every fixer protects it.
Legume nodules: leghaemoglobin binds O₂ with high affinity, keeping free O₂ low while delivering it to bacteroid respiration.
Filamentous cyanobacteria such as Anabaena: heterocysts lack photosystem II, so they evolve no O₂, and have thick envelopes.
Azotobacter: a very high respiratory rate consumes O₂ before it reaches the enzyme (respiratory protection).

Ammonia enters amino acids by two routes. Glutamine synthetase (GS) adds NH₄⁺ to glutamate using ATP, and glutamate synthase (GOGAT) transfers the amide group of glutamine to α-ketoglutarate, giving two glutamate. GS has a high affinity for NH₄⁺, so the GS–GOGAT pair is the main route when ammonium is scarce. Glutamate dehydrogenase reductively aminates α-ketoglutarate, but its low affinity suits it only to high ammonium. Glutamate and glutamine then donate nitrogen to nearly every other nitrogenous compound.

2. Amino acid catabolism and the urea cycle

Most amino acids lose their α-amino group by transamination to α-ketoglutarate, giving glutamate and the corresponding α-keto acid. Aminotransferases use pyridoxal phosphate (PLP) from vitamin B₆: the amino acid forms a Schiff base with PLP, the amino group stays on the cofactor as pyridoxamine phosphate, and is then passed to the acceptor keto acid (a ping-pong mechanism). Serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) leak from damaged cells; ALT is the more liver-specific marker. Glutamate is then oxidatively deaminated in liver mitochondria by glutamate dehydrogenase (NAD⁺ or NADP⁺), which releases NH₄⁺ and is inhibited by GTP and activated by ADP. Muscle exports its nitrogen as alanine and, like most tissues, as glutamine.

The carbon skeletons end as pyruvate or TCA intermediates (glucogenic) or as acetyl-CoA and acetoacetate (ketogenic). Leucine and lysine are purely ketogenic; isoleucine, phenylalanine, tyrosine and tryptophan are both. The urea cycle of the liver spans two compartments:

Matrix: carbamoyl phosphate synthetase I (CPS I) makes carbamoyl phosphate from NH₄⁺, HCO₃⁻ and 2 ATP. It is inactive without its allosteric activator N-acetylglutamate, whose synthesis arginine stimulates, so a protein-rich meal speeds the cycle.
Matrix: ornithine transcarbamoylase (OTC) joins carbamoyl phosphate to ornithine, giving citrulline, which moves to the cytosol.
Cytosol: argininosuccinate synthetase condenses citrulline with aspartate, the source of the second nitrogen, using ATP → AMP + PPi.
Cytosol: argininosuccinate lyase releases arginine and fumarate, and arginase hydrolyses arginine to urea and ornithine, which returns to the matrix.

Each urea costs 3 ATP but 4 high-energy phosphate bonds. The fumarate links the urea cycle to the TCA cycle: fumarate → malate → oxaloacetate yields an NADH, and oxaloacetate is transaminated back to aspartate, which offsets part of the cost.

🎯 Hyperammonaemia: why it harms and how the block is located

Excess NH₄⁺ drives glutamate dehydrogenase and glutamine synthetase in the brain, draining α-ketoglutarate and accumulating glutamine in astrocytes, which swell. Every urea-cycle defect causes hyperammonaemia; the pattern of intermediates locates the block. In OTC deficiency (X-linked, the commonest) unused carbamoyl phosphate leaks to the cytosol and enters pyrimidine synthesis, so orotic acid appears in urine; CPS I deficiency gives no orotic aciduria. Citrulline is high when argininosuccinate synthetase is deficient. Treatment combines a low-protein diet, arginine and nitrogen scavengers: benzoate conjugates glycine to hippurate, and phenylbutyrate (as phenylacetate) conjugates glutamine, both excreted in urine.

3. Nucleotide metabolism: de novo synthesis, salvage and breakdown

Both families need PRPP (5-phosphoribosyl-1-pyrophosphate), made from ribose-5-phosphate and ATP. Purines are built on the sugar: glutamine-PRPP amidotransferase, the committed step inhibited by AMP, GMP and IMP, adds the first nitrogen, and the ring is assembled atom by atom to IMP. IMP branches to AMP (using GTP) and GMP (using ATP), a reciprocal arrangement that keeps the two balanced. Pyrimidines are built as a ring first and attached later: carbamoyl phosphate from cytosolic CPS II (using glutamine) condenses with aspartate (aspartate transcarbamoylase, ATCase); the ring closes and is oxidised to orotate, which is attached to PRPP (OMP) and decarboxylated to UMP. UTP is aminated to CTP. In E. coli ATCase is the control point, inhibited by CTP and activated by ATP; in animals CPS II is.

Where the ring atoms come from
Ring atoms	Source
Purine N-1	Aspartate (amino group)
Purine C-2 and C-8	N¹⁰-Formyltetrahydrofolate
Purine N-3 and N-9	Glutamine (amide nitrogen)
Purine C-4, C-5 and N-7	Glycine (whole molecule)
Purine C-6	CO₂ (as HCO₃⁻)
Pyrimidine N-1, C-4, C-5 and C-6	Aspartate
Pyrimidine C-2 and N-3	Carbamoyl phosphate (HCO₃⁻ and glutamine)

Ribonucleotide reductase converts NDPs to dNDPs and is allosterically tuned so that the four dNTPs are made in balance (dATP shuts down overall activity). Thymidylate synthase methylates dUMP to dTMP with N⁵,N¹⁰-methylene-tetrahydrofolate, which is oxidised to dihydrofolate; dihydrofolate reductase (DHFR) regenerates tetrahydrofolate. These are drug targets: 5-fluorouracil (as FdUMP) inactivates thymidylate synthase, methotrexate inhibits DHFR, trimethoprim inhibits bacterial DHFR and hydroxyurea inhibits ribonucleotide reductase. Salvage recycles free bases cheaply: HGPRT converts hypoxanthine and guanine (with PRPP) to IMP and GMP, and APRT does the same for adenine. Purines are degraded to uric acid (xanthine oxidase converts hypoxanthine to xanthine and xanthine to urate) because humans lack urate oxidase; pyrimidines give soluble β-alanine and β-aminoisobutyrate.

Gout: urate crystals in joints from overproduction or underexcretion; allopurinol inhibits xanthine oxidase.
Lesch–Nyhan syndrome: X-linked HGPRT deficiency. Unused PRPP accumulates and salvage products no longer feed back, so de novo synthesis and uric acid rise; self-injury and dystonia accompany the hyperuricaemia.
Adenosine deaminase deficiency: dATP accumulates in lymphocytes and inhibits ribonucleotide reductase, causing severe combined immunodeficiency.
Hereditary orotic aciduria: UMP synthase deficiency; megaloblastic anaemia that B₁₂ or folate does not cure, treated with uridine.

4. Photosynthesis: the light reactions and photophosphorylation

The thylakoid membrane carries two photosystems in series (the Z scheme). Photosystem II (reaction centre P680) uses light to take electrons from water at its oxygen-evolving Mn₄CaO₅ cluster: 2 H₂O → O₂ + 4 H⁺ + 4 e⁻, the protons being released into the lumen. Electrons pass through pheophytin and the quinones QA and QB to the plastoquinone pool, then through the cytochrome b₆f complex, which moves further protons into the lumen, to the copper protein plastocyanin. Photosystem I (P700) re-energises them and passes them through ferredoxin to ferredoxin–NADP⁺ reductase, which makes NADPH in the stroma.

Photophosphorylation is chemiosmotic, like oxidative phosphorylation but with the topology reversed: protons accumulate in the thylakoid lumen and flow out to the stroma through CF₀CF₁ ATP synthase, whose head faces the stroma. In non-cyclic flow, from water to NADP⁺, both photosystems work and ATP, NADPH and O₂ are made. In cyclic flow, ferredoxin returns electrons to the cytochrome b₆f complex and back to P700; only photosystem I works, and ATP is made with no NADPH and no O₂. Cyclic flow tops up ATP, because the Calvin cycle needs 3 ATP for every 2 NADPH.

Emerson enhancement: far-red light (beyond 680 nm) alone drives photosynthesis poorly (the red drop), but adding shorter-wavelength red light raises the rate above the sum of the two beams used separately, showing that two photosystems act in series.
Jagendorf's acid-bath experiment: thylakoids equilibrated at pH 4 in the dark and transferred to pH 8 with ADP and Pi made ATP without light, showing that a proton gradient alone drives ATP synthesis.
DCMU (diuron) occupies the QB site of photosystem II, stopping O₂ evolution and NADP⁺ reduction while cyclic flow around photosystem I continues. Paraquat diverts electrons from photosystem I to O₂, making damaging superoxide.
Quantum requirement: moving one electron from water to NADP⁺ needs two photons, one per photosystem, so evolving one O₂ (4 e⁻) needs at least 8 photons.

5. The Calvin cycle and its regulation

The Calvin cycle in the stroma has three phases. Carboxylation: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) adds CO₂ to ribulose-1,5-bisphosphate (RuBP), giving two 3-phosphoglycerate; Calvin's ¹⁴CO₂ pulse experiments on Chlorella found 3-phosphoglycerate, labelled in its carboxyl carbon, as the first stable product. Reduction: phosphoglycerate kinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase convert 3-phosphoglycerate to triose phosphate, using 2 ATP and 2 NADPH per CO₂. Regeneration: aldolase, transketolase, sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase reshuffle five triose phosphates into three pentoses, and phosphoribulokinase spends one more ATP per CO₂ to remake RuBP.

Calvin cycle stoichiometry
Output	CO₂ fixed	ATP	NADPH
Per CO₂	1	3	2
One triose phosphate exported	3	9	6
One hexose	6	18	12

Light switches the cycle on in several ways. Proton pumping into the lumen raises the stromal pH from about 7 to about 8, and Mg²⁺ moves into the stroma in exchange; both activate Rubisco and fructose-1,6-bisphosphatase. Reduced ferredoxin reduces thioredoxin, which breaks regulatory disulfide bonds and activates fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and phosphoribulokinase. Rubisco itself must be carbamylated: a CO₂ molecule distinct from the substrate forms a carbamate on an active-site lysine that then binds Mg²⁺, and Rubisco activase, using ATP, removes inhibitory sugar phosphates from the site. In the dark these enzymes switch off, so the cycle does not compete with the breakdown of sugar at night.

⚠️ Photorespiration

Rubisco also accepts O₂: RuBP + O₂ gives one 3-phosphoglycerate and one 2-phosphoglycolate. Salvaging glycolate through chloroplast, peroxisome and mitochondrion releases CO₂ and NH₃ and wastes ATP and reducing power. Oxygenation rises with temperature and with a low CO₂/O₂ ratio, which is why C₄ and CAM plants concentrate CO₂ around Rubisco.

6. Disorders of sugar, amino acid and lipid metabolism

Garrod's idea of inborn errors of metabolism, first argued from alkaptonuria, rests on one-step logic: a missing enzyme makes its substrate pile up (and spill into side routes) and its product run short. Read every disorder that way. Most are autosomal recessive; familial hypercholesterolaemia is dominant, and OTC deficiency, Lesch–Nyhan syndrome, G6PD deficiency and Fabry disease are X-linked. Diabetes mellitus, the commonest disorder of fuel metabolism, is acquired rather than inborn: without insulin action the liver behaves as if fasting while blood glucose is high, lipolysis floods it with fatty acids, and in type 1 diabetes ketone bodies can accumulate to ketoacidosis.

Disorders of sugar metabolism
Disorder	Defect	Key consequences
Classic galactosaemia	Galactose-1-phosphate uridylyltransferase	Galactose-1-phosphate accumulates: vomiting, liver damage, cataract (galactitol), intellectual disability; remove galactose from the diet
Hereditary fructose intolerance	Aldolase B	Fructose-1-phosphate traps Pi, ATP falls, glycogen breakdown and gluconeogenesis stall: hypoglycaemia after fructose or sucrose
Essential fructosuria	Fructokinase	Fructose appears in urine; harmless
Lactose intolerance	Intestinal lactase (non-persistence after weaning)	Colonic fermentation: bloating and osmotic diarrhoea
Von Gierke disease (glycogen storage type I)	Glucose-6-phosphatase	Fasting hypoglycaemia unresponsive to glucagon, enlarged liver, lactic acidosis, hyperuricaemia, hyperlipidaemia
Pompe disease (type II)	Lysosomal acid α-1,4-glucosidase	Glycogen stored in lysosomes; cardiomyopathy and muscle weakness
McArdle disease (type V)	Muscle glycogen phosphorylase	Exercise cramps and myoglobinuria; blood lactate fails to rise on exercise

Disorders of amino acid and lipid metabolism
Disorder	Defect	Key consequences
Phenylketonuria	Phenylalanine hydroxylase, or its cofactor tetrahydrobiopterin	Phenylalanine and phenylpyruvate accumulate, tyrosine becomes essential; intellectual disability unless a low-phenylalanine diet starts in infancy; found by newborn screening
Alkaptonuria	Homogentisate oxidase (homogentisate 1,2-dioxygenase)	Urine darkens on standing; ochronosis and arthritis in adults
Oculocutaneous albinism type 1	Tyrosinase	No melanin in skin, hair and eyes
Maple syrup urine disease	Branched-chain α-keto acid dehydrogenase	Leucine, isoleucine, valine and their keto acids accumulate; sweet-smelling urine, encephalopathy
Classic homocystinuria	Cystathionine β-synthase (needs PLP)	Homocysteine and methionine rise; lens dislocation, thrombosis; some forms respond to vitamin B₆
Familial hypercholesterolaemia	LDL receptor	High LDL cholesterol from birth, xanthomas, early coronary disease; statins inhibit HMG-CoA reductase
Lipoprotein lipase deficiency	Lipoprotein lipase (or its activator apoC-II)	Chylomicrons and triacylglycerol very high; pancreatitis, eruptive xanthomas
MCAD deficiency	Medium-chain acyl-CoA dehydrogenase	Hypoketotic hypoglycaemia on fasting; avoid fasting
Tay–Sachs disease	Hexosaminidase A	GM2 ganglioside in neurons; neurodegeneration, cherry-red macula
Gaucher disease	β-Glucocerebrosidase	Glucocerebroside in macrophages; enlarged liver and spleen, bone disease; enzyme replacement available
Fabry disease	α-Galactosidase A (X-linked)	Globotriaosylceramide storage; painful neuropathy, kidney failure

Key takeaways

Nitrogenase: N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂; it is O₂-sensitive and protected by leghaemoglobin, heterocysts or fast respiration; GS–GOGAT assimilates scarce ammonium.
Transamination (PLP) funnels amino groups to glutamate and glutamate dehydrogenase releases NH₄⁺; urea takes one N from NH₄⁺ through CPS I (which needs N-acetylglutamate) and one from aspartate, at 4 high-energy phosphates. Orotic aciduria separates OTC from CPS I deficiency.
Purines are built on PRPP up to IMP (ring atoms from glycine, glutamine, aspartate, formyl-THF and CO₂); pyrimidines are built as orotate and then attached to PRPP. HGPRT loss causes Lesch–Nyhan syndrome; DHFR, thymidylate synthase and xanthine oxidase are drug targets.
Non-cyclic flow makes ATP, NADPH and O₂ (at least 8 photons per O₂); cyclic flow around photosystem I makes only ATP; DCMU blocks photosystem II; a proton gradient alone drives ATP synthesis.
The Calvin cycle uses 3 ATP and 2 NADPH per CO₂ (9 and 6 per triose phosphate, 18 and 12 per hexose); light activates it through stromal pH and Mg²⁺, thioredoxin and Rubisco carbamylation; oxygenation causes photorespiration.
Read each inborn error as substrate up, product down: galactosaemia, fructose intolerance, von Gierke, McArdle, phenylketonuria, alkaptonuria, maple syrup urine disease, urea-cycle defects, familial hypercholesterolaemia and the sphingolipidoses.

Practice questions (18)

Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.

What is the role of leghaemoglobin in the root nodules of legumes?
1. It transfers electrons from ferredoxin to the Fe protein of nitrogenase
2. It forms the iron–molybdenum cofactor at the active site of nitrogenase
3. It binds O₂ tightly, keeping free O₂ low while supplying bacteroid respiration
4. It stores the fixed ammonia as a haem complex until the host plant needs it
Show answer
Answer: C — It binds O₂ tightly, keeping free O₂ low while supplying bacteroid respiration
Nitrogenase is irreversibly inactivated by O₂, yet the bacteroids need O₂ for the respiration that supplies the ATP nitrogenase consumes. Leghaemoglobin, with a very high O₂ affinity, buffers free O₂ at a low concentration and delivers it to the bacteroids' terminal oxidases. Electrons reach the Fe protein from ferredoxin or flavodoxin, and the FeMo cofactor belongs to the MoFe protein, not to a globin.
When ammonium is scarce, bacteria and plants assimilate it mainly through glutamine synthetase and glutamate synthase (GOGAT) rather than through glutamate dehydrogenase. Why?
1. Glutamate dehydrogenase works only in the deamination direction and cannot add ammonia to α-ketoglutarate
2. Glutamine synthetase has a much higher affinity for NH₄⁺, and its ATP hydrolysis drives assimilation at low concentration
3. Glutamate synthase fixes atmospheric N₂ directly into glutamate without any need for nitrogenase
4. Glutamine synthetase uses NADPH, which is far more plentiful than ATP when ammonium is limiting
Show answer
Answer: B — Glutamine synthetase has a much higher affinity for NH₄⁺, and its ATP hydrolysis drives assimilation at low concentration
GS couples the amidation of glutamate to ATP hydrolysis and has a low Km for NH₄⁺, so it captures ammonium at low concentration; GOGAT then moves the amide nitrogen to α-ketoglutarate, giving two glutamate. Glutamate dehydrogenase is reversible and can aminate α-ketoglutarate, but its high Km for NH₄⁺ makes it effective only when ammonium is abundant. GS uses ATP, not NADPH, and neither enzyme touches N₂.
All aminotransferases require pyridoxal phosphate. What does the cofactor do in the reaction?
1. It accepts the amino group as free NH₄⁺, which is then handed directly to carbamoyl phosphate synthetase
2. It carries a one-carbon unit from serine to the α-keto acid acceptor of the reaction
3. It oxidises the amino acid with NAD⁺ to an imine, which water then hydrolyses to the keto acid
4. It forms a Schiff base with the amino acid and holds the amino group as pyridoxamine phosphate
Show answer
Answer: D — It forms a Schiff base with the amino acid and holds the amino group as pyridoxamine phosphate
The aldehyde of PLP forms a Schiff base with the incoming amino acid; the pyridine ring acts as an electron sink, the amino group stays on the cofactor as pyridoxamine phosphate and the α-keto acid leaves. In the second half of the ping-pong reaction the amino group is passed to the acceptor keto acid, usually α-ketoglutarate. Transamination releases no free NH₄⁺; that happens in the NAD⁺-dependent oxidative deamination by glutamate dehydrogenase, which a distractor describes. One-carbon transfer is the job of tetrahydrofolate.
Two newborns have hyperammonaemia with low plasma citrulline. One excretes large amounts of orotic acid in urine; the other does not. Which enzyme is most likely deficient in the infant with orotic aciduria?
1. Ornithine transcarbamoylase, because unused carbamoyl phosphate leaks to the cytosol and enters pyrimidine synthesis
2. Carbamoyl phosphate synthetase I, because the lack of carbamoyl phosphate overstimulates cytosolic pyrimidine synthesis
3. Argininosuccinate synthetase, because citrulline that cannot be used is converted into orotic acid by the liver
4. Arginase, because ornithine runs short and carbamoyl phosphate is turned into orotate inside the mitochondrion
Show answer
Answer: A — Ornithine transcarbamoylase, because unused carbamoyl phosphate leaks to the cytosol and enters pyrimidine synthesis
Low citrulline places the block before citrulline is made: CPS I or OTC. In OTC deficiency carbamoyl phosphate is still made but cannot react with ornithine; it spills into the cytosol, where aspartate transcarbamoylase uses it, so orotate is overproduced. In CPS I deficiency no carbamoyl phosphate is made, so there is no orotic aciduria: that is the other infant. Argininosuccinate synthetase deficiency raises citrulline, and arginase deficiency raises arginine, so neither fits.
Which of the following statements about the urea cycle are correct?
1. N-Acetylglutamate is an essential allosteric activator of carbamoyl phosphate synthetase I
2. Both nitrogen atoms of urea come from free NH₄⁺ that enters through carbamoyl phosphate
3. Argininosuccinate synthetase cleaves ATP to AMP and pyrophosphate
4. Ornithine transcarbamoylase and arginase both act in the mitochondrial matrix of hepatocytes
Show answer
Answer: A — N-Acetylglutamate is an essential allosteric activator of carbamoyl phosphate synthetase I; C — Argininosuccinate synthetase cleaves ATP to AMP and pyrophosphate
CPS I is virtually inactive without N-acetylglutamate, whose synthesis arginine stimulates. Argininosuccinate synthetase activates citrulline as a citrullyl-AMP intermediate and releases PPi, which is hydrolysed, so this step costs two high-energy bonds. One urea nitrogen comes from NH₄⁺ through carbamoyl phosphate and the other from aspartate. OTC is mitochondrial, but arginase acts in the cytosol.
Methotrexate is used against rapidly dividing cancer cells. Its main action is to
1. inhibit dihydrofolate reductase, so tetrahydrofolate is not regenerated after thymidylate synthesis
2. inactivate thymidylate synthase covalently after its conversion to a fluorinated deoxynucleotide
3. inhibit ribonucleotide reductase by scavenging the tyrosyl radical that its catalysis requires
4. inhibit xanthine oxidase, so that purine bases cannot be degraded and accumulate inside cells
Show answer
Answer: A — inhibit dihydrofolate reductase, so tetrahydrofolate is not regenerated after thymidylate synthesis
Thymidylate synthase oxidises N⁵,N¹⁰-methylene-tetrahydrofolate to dihydrofolate while making dTMP, and dihydrofolate reductase must reduce it back. Methotrexate, a folate analogue, binds DHFR very tightly, so tetrahydrofolate runs out and dTMP and purine synthesis stop in dividing cells. The fluorinated nucleotide describes 5-fluorouracil (FdUMP), radical scavenging describes hydroxyurea, and xanthine oxidase is the target of allopurinol.
A boy with self-injurious behaviour and gouty arthritis has a raised serum uric acid. His cultured cells contain high PRPP and show an increased rate of de novo purine synthesis. Which defect best accounts for all these findings?
1. Loss of xanthine oxidase activity, so that hypoxanthine accumulates and drives de novo synthesis
2. Deficiency of adenosine deaminase, so that deoxyadenosine nucleotides accumulate in his lymphocytes
3. Deficiency of hypoxanthine-guanine phosphoribosyltransferase, so salvage no longer consumes PRPP
4. A PRPP synthetase that has lost activity, so purine nucleotides cannot be made by either route
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Answer: C — Deficiency of hypoxanthine-guanine phosphoribosyltransferase, so salvage no longer consumes PRPP
HGPRT normally uses PRPP to salvage hypoxanthine and guanine. When it is missing, PRPP accumulates and stimulates glutamine-PRPP amidotransferase, while less IMP and GMP are available for feedback; de novo synthesis rises and the surplus purines are degraded to uric acid. Loss of xanthine oxidase would lower uric acid, ADA deficiency causes immunodeficiency without hyperuricaemia, and an inactive PRPP synthetase would lower PRPP, the opposite of what was found.
Which electron carrier transfers electrons from the cytochrome b₆f complex to photosystem I?
1. Plastoquinone, the mobile carrier that diffuses within the membrane
2. Ferredoxin, the soluble iron–sulfur protein of the stroma
3. Pheophytin, the first electron acceptor within photosystem II
4. Plastocyanin, a copper protein in the thylakoid lumen
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Answer: D — Plastocyanin, a copper protein in the thylakoid lumen
Plastocyanin, a small copper protein, shuttles electrons in the lumen from cytochrome f of the b₆f complex to P700⁺. Plastoquinone carries electrons from photosystem II to the b₆f complex, pheophytin is the primary acceptor inside photosystem II, and ferredoxin lies on the stromal side, beyond photosystem I.
Illuminated chloroplasts supplied with ADP, Pi and NADP⁺ are treated with DCMU. Which observation is expected?
1. O₂ evolution continues, but NADP⁺ reduction stops because DCMU blocks ferredoxin–NADP⁺ reductase
2. O₂ evolution and NADP⁺ reduction stop, but cyclic photophosphorylation can still make ATP
3. All ATP synthesis stops at once, because DCMU makes the thylakoid membrane leaky to protons
4. NADP⁺ reduction continues with electrons that photosystem I removes from water by itself
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Answer: B — O₂ evolution and NADP⁺ reduction stop, but cyclic photophosphorylation can still make ATP
DCMU binds the QB site of the D1 protein of photosystem II and blocks electron transfer to plastoquinone. Non-cyclic flow from water therefore stops, and with it O₂ evolution and NADPH formation. Cyclic flow around photosystem I does not involve photosystem II, so it can still move protons through the cytochrome b₆f complex and make ATP. DCMU is not an uncoupler, and photosystem I cannot oxidise water.
Which of the following statements about the Calvin cycle and its regulation are correct?
1. Illumination raises stromal pH and Mg²⁺, both of which activate Rubisco and fructose-1,6-bisphosphatase
2. Reduced thioredoxin activates fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and phosphoribulokinase in the light
3. The CO₂ that carbamylates the active-site lysine of Rubisco is the same molecule that is fixed into 3-phosphoglycerate
4. The oxygenase reaction of Rubisco gives two molecules of 3-phosphoglycerate for each O₂ consumed
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Answer: A — Illumination raises stromal pH and Mg²⁺, both of which activate Rubisco and fructose-1,6-bisphosphatase; B — Reduced thioredoxin activates fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and phosphoribulokinase in the light
Proton pumping into the lumen makes the stroma alkaline (about pH 8), and Mg²⁺ enters the stroma in exchange; Rubisco and stromal FBPase work best under these conditions. Ferredoxin–thioredoxin reductase reduces thioredoxin, which reduces regulatory disulfides of FBPase, SBPase and phosphoribulokinase. The activator CO₂ forms a carbamate with the lysine and stays bound with Mg²⁺, while a separate substrate CO₂ is fixed. Oxygenation gives one 3-phosphoglycerate and one 2-phosphoglycolate.
An infant thrives on breast milk but develops vomiting, hypoglycaemia and an enlarged liver soon after fruit juice and sucrose are added to the diet. Which enzyme is most likely deficient?
1. Fructokinase, which phosphorylates fructose to fructose-1-phosphate in the liver
2. Galactose-1-phosphate uridylyltransferase, which converts galactose to a glucose derivative
3. Aldolase B, which cleaves fructose-1-phosphate into two three-carbon products
4. Glucose-6-phosphatase, which releases free glucose from the liver between meals
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Answer: C — Aldolase B, which cleaves fructose-1-phosphate into two three-carbon products
In hereditary fructose intolerance fructokinase still makes fructose-1-phosphate but aldolase B cannot cleave it, so phosphate is trapped, ATP falls, and glycogen breakdown and gluconeogenesis are inhibited, giving hypoglycaemia after fructose or sucrose. Fructokinase deficiency (essential fructosuria) is harmless because fructose is never trapped as a phosphate. Galactosaemia would appear on breast milk, which contains lactose, and glucose-6-phosphatase deficiency causes fasting hypoglycaemia regardless of fructose.
Which of the following statements about inherited disorders of amino acid and lipid metabolism are correct?
1. In classic phenylketonuria, tyrosine becomes a dietary essential amino acid
2. In alkaptonuria, homogentisate oxidase is deficient and the urine darkens on standing in air
3. Maple syrup urine disease is a defect in the breakdown of the aromatic amino acids
4. In Tay–Sachs disease, hexosaminidase A is deficient and GM2 ganglioside accumulates in neurons
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Answer: A — In classic phenylketonuria, tyrosine becomes a dietary essential amino acid; B — In alkaptonuria, homogentisate oxidase is deficient and the urine darkens on standing in air; D — In Tay–Sachs disease, hexosaminidase A is deficient and GM2 ganglioside accumulates in neurons
With phenylalanine hydroxylase deficient, tyrosine is no longer made from phenylalanine and must come from the diet. Homogentisate that cannot be cleaved is excreted and oxidises in air to a dark pigment. Hexosaminidase A removes the terminal N-acetylgalactosamine of GM2, so GM2 accumulates without it. Maple syrup urine disease is a defect of the branched-chain α-keto acid dehydrogenase, so leucine, isoleucine and valine and their keto acids accumulate; the aromatic amino acids are not involved.
Patient X has fasting hypoglycaemia, an enlarged liver, lactic acidosis and no rise in blood glucose after glucagon. Patient Y has muscle cramps during exercise, and his blood lactate does not rise during an ischaemic forearm exercise test. Which deficiencies explain X and Y respectively?
1. Muscle glycogen phosphorylase in X; glucose-6-phosphatase in Y
2. Glucose-6-phosphatase in X; lysosomal acid α-glucosidase in Y
3. Lysosomal acid α-glucosidase in X; liver glycogen phosphorylase in Y
4. Glucose-6-phosphatase in X; muscle glycogen phosphorylase in Y
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Answer: D — Glucose-6-phosphatase in X; muscle glycogen phosphorylase in Y
In von Gierke disease (glucose-6-phosphatase deficiency) glucose-6-phosphate made from glycogen or by gluconeogenesis cannot be released as glucose, so glucagon fails to raise blood glucose, and the trapped glucose-6-phosphate is pushed through glycolysis to lactate. In McArdle disease muscle cannot break down its glycogen, so exercising muscle runs short of fuel and makes no extra lactate. Lysosomal acid α-glucosidase deficiency (Pompe disease) stores glycogen in lysosomes and causes cardiomyopathy and weakness, not an absent lactate rise, and liver phosphorylase does not act in muscle.
Taking the nitrogenase reaction as N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 Pi, how many ATP are hydrolysed per molecule of NH₃ formed?
Numerical answer — type the value.
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Answer: 8
16 ATP give 2 NH₃, so 16 / 2 = 8 ATP per NH₃ (2 ATP per electron, with the 8 electrons, including the 2 lost to H₂, shared over two NH₃). Answering 16 counts per N₂ rather than per NH₃, and answering 6 ignores the obligatory H₂ (6 electrons per N₂).
Count each phosphoanhydride bond cleaved in the urea cycle as one ATP equivalent (pyrophosphate released is hydrolysed). Assume that the fumarate released is converted to oxaloacetate in a way that yields one NADH, worth 2.5 ATP, and ignore all other energy changes. What is the net ATP cost of producing one molecule of urea, to one decimal place?
Numerical answer — type the value.
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Answer: 1.5
CPS I uses 2 ATP → 2 ADP (2 bonds). Argininosuccinate synthetase uses ATP → AMP + PPi, and PPi is hydrolysed (2 bonds). Gross cost 4. Fumarate → malate → oxaloacetate gives one NADH worth 2.5. Net 4 − 2.5 = 1.5. Counting 3 ATP molecules instead of 4 bonds gives 0.5.
In de novo purine synthesis, how many atoms of the purine ring system are contributed by glycine?
Numerical answer — type the value.
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Answer: 3
Glycine is incorporated whole and supplies C-4, C-5 and N-7: three atoms. The other ring atoms are N-1 from aspartate, C-2 and C-8 from N¹⁰-formyltetrahydrofolate, N-3 and N-9 from the amide nitrogen of glutamine, and C-6 from CO₂, nine atoms in all. Answering 2 counts only glycine's carbons.
A leaf fixes 6 CO₂ into one hexose by the Calvin cycle. Assume that all the NADPH comes from non-cyclic electron flow, that moving one electron from water to NADP⁺ requires two photons (one at each photosystem), and ignore any photons used to make extra ATP. What is the minimum number of photons absorbed?
Numerical answer — type the value.
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Answer: 48
Each CO₂ needs 2 NADPH = 4 electrons; 4 electrons × 2 photons = 8 photons per CO₂, the same as the 8 photons per O₂ evolved (each O₂ releases 4 electrons). For 6 CO₂: 6 × 8 = 48. Counting one photon per electron gives 24, and one photon per NADPH gives 12.
How many ATP molecules does the Calvin cycle consume to produce one molecule of glyceraldehyde-3-phosphate for export from the chloroplast?
Numerical answer — type the value.
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Answer: 9
Exporting one triose phosphate requires the net fixation of 3 CO₂. Each CO₂ costs 2 ATP at phosphoglycerate kinase (two 3-phosphoglycerate reduced) and 1 ATP at phosphoribulokinase, so 3 ATP per CO₂ and 9 per triose, along with 6 NADPH. Answering 3 gives the cost per CO₂, and 18 the cost per hexose.