EXAM STRATEGY & IMPORTANT QUESTIONS GUIDE

Carbohydrates:
Carbohydrates are polyhydroxy aldehydes or ketones (or compounds that yield them on hydrolysis) with the general formula (CH₂O)ₙ.
Classification:
Monosaccharides 🔊 cannot be hydrolysed further and range from trioses (3 C) to heptoses (7 C); common examples are glucose, fructose, galactose and ribose.
Disaccharides consist of two monosaccharide units: sucrose (glucose + fructose), lactose (galactose + glucose) and maltose (glucose + glucose).
Oligosaccharides contain 3 to 10 units, while polysaccharides contain many units and include starch, glycogen and cellulose.
Biological roles: carbohydrates serve as an energy source (4 kcal/g), as energy stores (glycogen in liver and muscle), as structural components (cellulose in plants and chitin in fungi), in signalling (glycoproteins), and as the ribose and deoxyribose of nucleic acids.

Lipids:
Lipids are a heterogeneous group of compounds that are insoluble in water but soluble in organic solvents.
Bloor's classification:
Simple lipids are esters of fatty acids with an alcohol and include fats, oils (triglycerides) and waxes.
Compound lipids have an additional group — phospholipids carry a phosphate, glycolipids a carbohydrate, and lipoproteins a protein.
Derived lipids are the products of hydrolysis and include fatty acids, glycerol, cholesterol, steroid hormones and the fat-soluble vitamins A, D, E and K.
Biological roles: lipids are the densest energy store (9 kcal/g), form the phospholipid bilayer of cell membranes, provide insulation and cushioning, serve as steroid hormones and signal molecules (prostaglandins), and are the basis of lipoprotein transport.

Proteins:
Proteins are polymers of the 20 standard amino acids joined by peptide bonds.
They have a four-level structural hierarchy. The primary structure is the amino-acid sequence; the secondary structure comprises local elements such as α-helix and β-sheet held by hydrogen bonds; the tertiary structure is the three-dimensional folding maintained by hydrogen, disulphide, ionic and hydrophobic interactions; and the quaternary structure is the assembly of two or more polypeptide subunits (for example haemoglobin α₂β₂).
Proteins are classified functionally (enzymes, transport proteins, structural proteins, contractile proteins, hormones and defence proteins), by solubility (albumins and globulins) and by shape (fibrous versus globular).
Biological roles: catalysis (enzymes), transport (haemoglobin, albumin), structural support (collagen, keratin), movement (actin and myosin), regulation (hormones), immunity (antibodies) and maintenance of homeostasis.

Nucleic Acids:
Nucleic acids are polymers of nucleotides.
DNA (deoxyribonucleic acid) contains bases A, T, G and C, exists as a double helix (Watson–Crick), and stores the genetic information.
RNA (ribonucleic acid) contains A, U, G and C, is mainly single-stranded, and exists as three principal types: mRNA, tRNA and rRNA.
Biological roles: storage of genetic information (DNA), information transfer (mRNA), protein synthesis (tRNA and rRNA), regulation of gene expression (miRNA and siRNA), and catalysis (ribozymes).

The 20 Standard Amino Acids:
The nine essential amino acids cannot be synthesised in the human body and must come from the diet; they are remembered by the mnemonic "PVT TIM HaLL" — phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine and lysine.
The eleven non-essential amino acids are synthesised in the body: alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, proline, serine, tyrosine and arginine.
On the basis of their side chains, amino acids are grouped as non-polar, polar, acidic or basic; the three aromatic members are phenylalanine, tyrosine and tryptophan.

Free Energy (G) and Related Terms:
The Gibbs free energy (G) is the energy available to do useful work at constant temperature and pressure and is defined by:
ΔG = ΔH − TΔS where ΔH is the change in enthalpy (heat), ΔS is the change in entropy (disorder) and T is the absolute temperature.
An exergonic reaction has ΔG < 0 and proceeds spontaneously with release of energy; an endergonic reaction has ΔG > 0, is non-spontaneous and requires energy input; a reaction at equilibrium has ΔG = 0. The standard free-energy change (ΔG°′) is measured at pH 7, 25 °C and 1 M concentration. Endergonic reactions are driven forward by coupling them to exergonic ones, most often ATP hydrolysis.

Redox Potential (E°):
The redox potential measures the tendency of a compound to gain or lose electrons. A more negative E° denotes a stronger reductant and a more positive E° a stronger oxidant. The two are related through ΔG° = −nFΔE°, the basis of biological oxidation in the electron-transport chain (NAD → FMN → CoQ → cytochromes → O₂).

Energy-Rich Compounds:
Compounds whose hydrolysis releases more than 7 kcal/mol are termed "high-energy compounds". They are classified by the type of high-energy bond as pyrophosphates (ATP, ADP, GTP, UTP), acyl phosphates (1,3-bisphosphoglycerate), enol phosphates (phosphoenolpyruvate), guanidino phosphates (creatine phosphate) and thioesters (acetyl-CoA, succinyl-CoA). Typical ΔG°′ values (in kcal/mol) are PEP −14.8, 1,3-BPG −11.8, creatine phosphate −10.3, ATP −7.3 and glucose-6-phosphate only −3.3.

Biological Significance of ATP:
ATP consists of adenine, ribose and three phosphate groups (α, β and γ). Hydrolysis of the terminal γ-phosphate bond releases about 7.3 kcal/mol.
Its biological roles are the following. It acts as the universal energy currency, driving endergonic reactions by coupled hydrolysis (ATP → ADP + Pi). It phosphorylates substrates (for example glucose to glucose-6-phosphate). It powers active transport (Na⁺/K⁺ pump, Ca²⁺ pump and H⁺/K⁺ pump). It drives muscle contraction at the myosin head. It supplies the energy for biosynthesis of proteins, nucleic acids and polysaccharides. It is the precursor of signalling molecules such as cAMP. It provides the energy for mechanical work of cilia and flagella.
The turnover of ATP is enormous: about 50 kg per day is cycled, although the body contains only about 50 g at any moment.

Biological Significance of cAMP:
Synthesis: cAMP is formed from ATP by adenylyl cyclase 🔊, which is activated by the Gsα subunit of a G-protein-coupled receptor.
Hydrolysis: cAMP is hydrolysed by phosphodiesterase to inactive 5′-AMP; the methylxanthines caffeine and theophylline inhibit this enzyme.
Sutherland's second-messenger concept: a hormone (first messenger) binds its receptor, activating adenylyl cyclase to produce cAMP (the second messenger); cAMP then activates PKA, which phosphorylates target proteins to produce the cellular response.
Examples of cAMP-mediated effects are adrenaline acting through β-adrenoceptors to cause hepatic glycogenolysis, glucagon raising blood glucose, ACTH stimulating cortisol release from the adrenal cortex, TSH stimulating release of T₃ and T₄ from the thyroid, and ADH promoting water retention in the kidney.

Overview:
Glycolysis takes place in the cytosol of every cell. One molecule of glucose (6 carbons) is converted into two molecules of pyruvate (3 carbons each), with a net production of 2 ATP and 2 NADH. The sequence has 10 enzymatic steps, organised into a preparatory (investment) phase that consumes 2 ATP, and a payoff phase that generates 4 ATP and 2 NADH.

Pathway (10 Steps):
Preparatory Phase (uses 2 ATP):
1. Glucose → Glucose-6-P by hexokinase / glucokinase (uses 1 ATP; irreversible).
2. Glucose-6-P → Fructose-6-P by phosphoglucose isomerase.
3. Fructose-6-P → Fructose-1,6-bisphosphate by phosphofructokinase-1 (PFK-1) (uses 1 ATP; rate-limiting; irreversible).
4. Fructose-1,6-BP → 2 × Triose phosphates (DHAP + G3P) by aldolase.
5. DHAP ↔ G3P by triose phosphate isomerase.
Payoff Phase (generates 4 ATP + 2 NADH) — per G3P, doubled for 2 molecules:
6. G3P + NAD⁺ + Pi → 1,3-bisphosphoglycerate + NADH by G3P dehydrogenase.
7. 1,3-BPG → 3-phosphoglycerate + ATP by phosphoglycerate kinase (substrate-level phosphorylation).
8. 3-PG → 2-PG by phosphoglycerate mutase.
9. 2-PG → Phosphoenolpyruvate (PEP) + H₂O by enolase.
10. PEP → Pyruvate + ATP by pyruvate kinase (substrate-level phosphorylation; irreversible).

Energetics:
Gross: 4 ATP (steps 7+10 ×2) + 2 NADH.
Used: 2 ATP (steps 1+3).
Net Glycolysis: 2 ATP + 2 NADH + 2 pyruvate.
If aerobic: 2 NADH → ETC → 5 ATP (if malate-aspartate shuttle) or 3 ATP (if glycerol-3-P shuttle). Plus pyruvate → acetyl-CoA → TCA = more ATP. Total ~30-32 ATP per glucose.
If anaerobic: pyruvate → lactate by LDH (regenerates NAD⁺); only 2 ATP.

Regulation (3 irreversible steps):
Hexokinase — inhibited by G-6-P.
PFK-1 (major) — inhibited by ATP, citrate; activated by AMP, F-2,6-BP.
Pyruvate kinase — inhibited by ATP, alanine; activated by F-1,6-BP.

Significance:
1. Provides energy rapidly, with/without O₂.
2. Supplies pyruvate → TCA cycle → fatty acid synthesis → cholesterol.
3. Supplies glycerol-P (for triglyceride synthesis) via DHAP.
4. Generates NADH for biosynthesis.
5. Only energy source for mature RBCs (no mitochondria) + anaerobic tissues.
6. Supports intense muscle activity (anaerobic → lactate).

Overview:
• Site: mitochondrial matrix.
• Input per turn: 1 acetyl-CoA (2 C from pyruvate, or fatty acid, or amino acid).
• Output per turn: 2 CO₂ + 3 NADH + 1 FADH₂ + 1 GTP + 1 CoA-SH.
• Final common pathway of CHO, lipid + protein oxidation.
• Total 8 steps.

Entry — Pyruvate → Acetyl-CoA:
Pyruvate (3C) + NAD⁺ + CoA-SH → Acetyl-CoA + CO₂ + NADH by Pyruvate dehydrogenase complex (3 enzymes, 5 coenzymes: TPP, lipoamide, FAD, NAD, CoA).

8 Steps of TCA Cycle:
1. Acetyl-CoA (2C) + Oxaloacetate (4C) → Citrate (6C) + CoA-SH by citrate synthase.
2. Citrate → Isocitrate by aconitase (via cis-aconitate).
3. Isocitrate → α-ketoglutarate + CO₂ + NADH by isocitrate dehydrogenase (rate-limiting; allosteric).
4. α-KG (5C) → Succinyl-CoA + CO₂ + NADH by α-KG dehydrogenase (similar to PDH).
5. Succinyl-CoA → Succinate + GTP by succinyl-CoA synthetase (substrate-level phosphorylation).
6. Succinate → Fumarate + FADH₂ by succinate dehydrogenase (only membrane-bound enzyme; part of Complex II of ETC).
7. Fumarate + H₂O → Malate by fumarase.
8. Malate + NAD⁺ → Oxaloacetate + NADH by malate dehydrogenase.

Energetics — Per Acetyl-CoA:

Item	Count	ATP equivalent (via ETC)
NADH	3	3 × 2.5 = 7.5
FADH₂	1	1 × 1.5 = 1.5
GTP	1	1
Total per acetyl-CoA		10 ATP

Total ATP per glucose (glycolysis + 2× PDH + 2× TCA):
Glycolysis 2 ATP + 2 NADH → 7; PDH 2 NADH → 5; TCA 20 → total = ~30-32 ATP per glucose (aerobic).

Regulation:
Citrate synthase — inhibited by ATP, citrate, succinyl-CoA.
Isocitrate dehydrogenase — main regulator; inhibited by ATP, NADH; activated by ADP, Ca²⁺.
α-KG dehydrogenase — inhibited by NADH, succinyl-CoA, ATP; activated by Ca²⁺.

Significance:
1. Central energy-producing pathway (aerobic).
2. Amphibolic — both catabolic + anabolic: supplies precursors for biosynthesis (α-KG → glutamate/aa; OAA → aspartate; succinyl-CoA → haem; citrate → fatty acids).
3. Provides CO₂ for urea synthesis.
4. Produces reducing equivalents (NADH, FADH₂) for ETC.
5. Links CHO, lipid + protein metabolism (final common pathway).
Anaplerotic reactions replenish TCA intermediates (pyruvate → OAA by pyruvate carboxylase).

HMP Shunt — Outline:
• Site: cytosol of liver, adipose, RBC, mammary gland, adrenal cortex, gonads.
• Two phases:
  Oxidative phase — G-6-P → 6-phosphogluconolactone → 6-phosphogluconate → Ribulose-5-P + 2 NADPH + CO₂. Rate-limiting enzyme: glucose-6-phosphate dehydrogenase (G6PD).
  Non-oxidative phase — interconversion: Ribulose-5-P ↔ Ribose-5-P + Xylulose-5-P; via transketolase (TPP-dependent) + transaldolase → can regenerate F-6-P + G-3-P (linking back to glycolysis).
Stoichiometry: 3 Glucose-6-P → 3 CO₂ + 6 NADPH + 2 F-6-P + 1 G-3-P.

Significance:
1. NADPH — for reductive biosynthesis (fatty acids, cholesterol, steroid hormones) + antioxidant (GSH regeneration in RBC).
2. Ribose-5-P — for nucleotide + nucleic acid synthesis.
3. No ATP produced / consumed.
4. Bacterial sensitivity / oxidative burst in phagocytes (NADPH oxidase).

G6PD Deficiency:
X-linked recessive; most common enzyme defect worldwide (400 million affected).
Consequence: ↓ NADPH in RBC → ↓ reduced glutathione (GSH) → cannot detoxify oxidants → RBC lyses.
Triggers (haemolytic crisis):
• Oxidant drugs — primaquine, sulphonamides, nitrofurantoin, dapsone.
• Fava beans (favism; Mediterranean variant).
• Infections.
• Naphthalene (moth balls).
Features: acute haemolytic anaemia, jaundice, dark urine (haemoglobinuria), Heinz bodies in RBC.
Diagnosis: enzyme assay; Methaemoglobin reduction test; fluorescent spot test.
Management: avoid triggers; transfusion if severe; folic acid supplement.

Electron Transport Chain:
Site: inner mitochondrial membrane. Four multi-protein complexes + 2 mobile carriers (CoQ / ubiquinone + cytochrome c).

Complex	Name	Function	H⁺ pumped?
I	NADH dehydrogenase (NADH-CoQ reductase)	NADH → FMN → Fe-S → CoQ	Yes (4 H⁺)
II	Succinate dehydrogenase	FADH₂ (from TCA step 6) → CoQ	No
III	Cytochrome bc₁ (CoQ-cyt c reductase)	CoQH₂ → cyt b → cyt c₁ → cyt c (mobile)	Yes (4 H⁺)
IV	Cytochrome c oxidase	cyt c → cyt a → cyt a₃ (Cu-haem) → O₂ → H₂O	Yes (2 H⁺)
V	ATP synthase (F₀F₁)	H⁺ flow drives ADP + Pi → ATP	— (uses gradient)

Final electron acceptor: ½ O₂ + 2 e⁻ + 2 H⁺ → H₂O.

Chemiosmotic Theory (Peter Mitchell, 1961):
• Electron transport pumps H⁺ from matrix to intermembrane space through complexes I, III, IV.
• Creates proton-motive force (PMF) — electrical + chemical gradient (pH difference).
• H⁺ flows back through F₀F₁ ATP synthase → rotation of γ-subunit → synthesis of ATP from ADP + Pi.
• P/O ratio: NADH → 2.5 ATP; FADH₂ → 1.5 ATP.

ATP Yield (per glucose, aerobic):
• Glycolysis — 2 ATP (net) + 2 NADH (cytosol) = 2 + 5 = 7.
• Pyruvate → Acetyl-CoA — 2 NADH = 5.
• TCA (2 rounds) — 6 NADH + 2 FADH₂ + 2 GTP = 15 + 3 + 2 = 20.
Total = 30-32 ATP per glucose.

Inhibitors of ETC (site-specific):

Inhibitor	Site / Complex
Rotenone, amytal (barbiturate), piericidin A	Complex I
Malonate (competitive), TTFA	Complex II
Antimycin A	Complex III
Cyanide (CN⁻), CO, azide (N₃⁻), H₂S	Complex IV
Oligomycin	ATP synthase (F₀ channel)

Uncouplers:
Dissipate the proton gradient without inhibiting electron flow → heat produced instead of ATP.
Examples:
• 2,4-Dinitrophenol (DNP) — classic chemical uncoupler; former anti-obesity drug (banned; fatal hyperthermia).
• Thermogenin (UCP-1) — natural uncoupler in brown adipose tissue; generates heat in neonates + hibernating animals.
• FCCP, CCCP (experimental).
• Aspirin in high dose, thyroxine (partial uncoupler).

Activation & Transport of Fatty Acid (Prior to β-Oxidation):
Step 1 — Activation (cytosol):
Fatty acid + CoA-SH + ATP → Fatty acyl-CoA + AMP + PPi (acyl-CoA synthetase / thiokinase; uses 2 ATP equivalents).
Step 2 — Transport into mitochondria: via carnitine shuttle (CPT-I outer + CPT-II inner membrane).
Acyl-CoA + carnitine → acyl-carnitine → crosses inner membrane → acyl-CoA inside matrix.

β-Oxidation — 4 Cyclic Steps (per cycle):
1. Dehydrogenation (FAD) — acyl-CoA → trans-Δ²-enoyl-CoA + FADH₂. Enzyme: acyl-CoA dehydrogenase.
2. Hydration — enoyl-CoA → β-hydroxyacyl-CoA. Enzyme: enoyl-CoA hydratase.
3. Dehydrogenation (NAD) — β-hydroxyacyl-CoA → β-ketoacyl-CoA + NADH. Enzyme: β-hydroxyacyl-CoA dehydrogenase.
4. Thiolytic cleavage — β-ketoacyl-CoA + CoA-SH → acyl-CoA (2 C shorter) + acetyl-CoA. Enzyme: thiolase.
Each cycle produces: 1 acetyl-CoA + 1 NADH + 1 FADH₂ + acyl-CoA (2C shorter).

Energetics — Palmitic Acid (C16):
7 cycles to completely degrade C16 → 8 acetyl-CoA + 7 NADH + 7 FADH₂.
Calculation:
• 7 NADH × 2.5 = 17.5 ATP
• 7 FADH₂ × 1.5 = 10.5 ATP
• 8 Acetyl-CoA × 10 (TCA) = 80 ATP
• Total = 108 ATP
• Subtract 2 ATP equivalents for activation
• Net = 106 ATP per palmitate.

Ketone Bodies — Formation (Ketogenesis):
Occurs in liver mitochondria when acetyl-CoA accumulates (starvation, diabetes, high-fat diet).
Pathway:
2 Acetyl-CoA → Acetoacetyl-CoA (thiolase reverse) →
+ Acetyl-CoA → HMG-CoA (β-hydroxy-β-methylglutaryl-CoA; HMG-CoA synthase) →
→ Acetoacetate (HMG-CoA lyase) →
→← β-hydroxybutyrate (reversible; β-HB dehydrogenase)
→ spontaneous decarboxylation → Acetone (fruity breath).
3 ketone bodies: Acetoacetate, β-hydroxybutyrate (main), Acetone (minor, volatile).
Utilisation (Ketolysis): extrahepatic tissues (brain, heart, muscle) convert β-HB → Acetoacetate → Acetoacetyl-CoA → 2 Acetyl-CoA → TCA cycle for energy.
Brain uses ketones during prolonged starvation (adaptation after ~3 days).

Ketoacidosis:
When ketone body production >> utilisation (e.g., uncontrolled DM Type 1; prolonged fasting; alcoholism).
Mechanism: ↑ lipolysis → ↑ FFA → ↑ β-oxidation → ↑ acetyl-CoA → ↑ ketones beyond utilisation → metabolic acidosis.
Features:
• Kussmaul breathing (deep rapid compensatory respiration).
• Fruity / acetone breath.
• Nausea, vomiting, abdominal pain.
• Dehydration (polyuria from glycosuria), hypotension.
• Altered consciousness → coma.
• Urine: strongly positive ketones (Rothera / nitroprusside test).
Treatment: insulin + fluids + K⁺ + HCO₃⁻ + treat precipitant.

General Reactions of Amino Acid Metabolism:
1. Transamination:
α-Amino acid + α-keto acid ⇌ α-keto acid + new α-amino acid.
Catalysed by transaminases (aminotransferases) using PLP (pyridoxal phosphate, Vit B6).
Examples: AST (Asp + α-KG ⇌ OAA + Glu), ALT (Ala + α-KG ⇌ Pyruvate + Glu).
Serum AST/ALT are classic liver-function markers.
2. Deamination:
Removal of amino group → free NH₃ + α-keto acid.
• Oxidative deamination (main) — glutamate + NAD⁺ → α-KG + NH₃ + NADH by glutamate dehydrogenase.
• Non-oxidative — serine / threonine dehydratases.
3. Decarboxylation:
Removal of -COOH group → biogenic amines.
Examples: Histidine → Histamine; Glutamate → GABA; Tyrosine → Dopamine → NA → Adrenaline; Tryptophan → Serotonin; Lysine → Cadaverine; Ornithine → Putrescine.
PLP-dependent.

Urea Cycle (Krebs-Henseleit, 1932):
Site: starts in mitochondria of hepatocytes, continues in cytosol.
Purpose: dispose of excess N (from protein catabolism) as non-toxic urea (excreted in urine).
5 enzymatic steps:
1. CPS-I (mitochondria): NH₃ + CO₂ + 2 ATP → carbamoyl phosphate. Rate-limiting; activated by N-acetyl glutamate.
2. Carbamoyl phosphate + Ornithine → Citrulline by Ornithine transcarbamoylase (OTC) (mito).
3. Citrulline exits to cytosol; + Aspartate + ATP → Argininosuccinate by argininosuccinate synthetase.
4. Argininosuccinate → Arginine + Fumarate by argininosuccinate lyase.
5. Arginine + H₂O → Urea + Ornithine by arginase. Ornithine recycled.
Net: 2 NH₃ + CO₂ + 3 ATP → Urea + 2 ADP + AMP + H₂O.
Fumarate re-enters TCA → OAA → aspartate (aspartate-arginine cycle).

Disorders of Urea Cycle:
Any defect → hyperammonaemia → toxic to brain (coma, convulsions, death).
Examples:
• CPS-I deficiency — neonatal hyperammonaemia.
• OTC deficiency — most common urea cycle disorder; X-linked; excess orotic acid.
• Citrullinaemia — ↓ argininosuccinate synthetase.
• Argininosuccinic aciduria — ↓ argininosuccinate lyase.
• Argininaemia — ↓ arginase.
Treatment: restrict protein intake; sodium benzoate / phenylacetate (conjugate N); arginine supplementation; liver transplant (severe).

Catabolic Pathway of Phe / Tyr:
Pathway:
Phenylalanine → Tyrosine (by phenylalanine hydroxylase; needs BH₄ + O₂) → p-hydroxyphenylpyruvate (transamination) → homogentisic acid → maleylacetoacetate → fumarylacetoacetate → Fumarate + Acetoacetate → TCA + ketogenesis.
Branching: Tyrosine also → DOPA → Dopamine → NA → Adrenaline (catecholamines) + Tyrosine → Melanin (in melanocytes, by tyrosinase) + Tyrosine → T₃, T₄ (thyroid hormone synthesis).

Disorders:
1. Phenylketonuria (Phe Hydroxylase deficiency):
• Autosomal recessive (AR).
• Phe accumulates, shunted to phenyl-pyruvate, phenyl-lactate, phenyl-acetate (urine).
• Clinical: mental retardation, fair skin (↓ melanin), mousy odour (phenylacetate), seizures, microcephaly.
• Diagnosis: Guthrie test (bacterial inhibition), tandem MS newborn screening, urine FeCl₃ test (green colour).
• Treatment: Phe-restricted diet from birth; avoid aspartame (contains Phe).

2. Albinism (Tyrosinase deficiency):
• Melanin not produced → very pale skin, white hair, pink-red iris, photophobia, ↑ skin cancer risk.
• Genetic: AR (tyrosinase-negative type) or AD (oculocutaneous).
• Management: sun protection, eye care.

3. Alkaptonuria (Homogentisic acid oxidase deficiency):
• First inborn error of metabolism described (Garrod, 1902).
• Homogentisic acid accumulates + excreted in urine → turns black on exposure to air / alkali.
• Ochronosis — dark pigment deposition in cartilage, tendons (dark ears, sclera); arthropathy in 3rd-4th decade.
• Benign in childhood; treatment: vitamin C, diet restriction, nitisinone (experimental).

4. Tyrosinaemia:
• Type I (most severe): ↓ fumarylacetoacetate hydrolase → toxic metabolites → liver/kidney failure.
• Type II: ↓ tyrosine aminotransferase → eye + skin lesions.
• Type III: ↓ 4-hydroxyphenylpyruvate dioxygenase.
• Treatment: Tyr / Phe restriction; nitisinone.

Catabolism of Haem:
Aged RBCs (120 d) phagocytosed by macrophages of RES (mainly spleen, liver, BM).
Steps:
Haem → Biliverdin (green) by haem oxygenase + releases Fe + CO →
→ Bilirubin (yellow) by biliverdin reductase + NADPH →
Bilirubin (unconjugated, indirect) is lipid-soluble, bound to albumin, transported to liver →
In liver → conjugated with 2 glucuronic acid by UDP-glucuronyl transferase → Conjugated (direct) bilirubin (water-soluble) →
Excreted in bile → intestine → urobilinogen (by gut bacteria) → partly reabsorbed (enterohepatic) or excreted as stercobilin (stool brown colour) + urobilin (urine yellow colour).

Jaundice:
Yellow discolouration of skin + sclera when serum bilirubin > 2 mg/dL (normal 0.2–1.0).
Classification:
1. Pre-hepatic / Haemolytic — excessive haemolysis → ↑ unconjugated bilirubin. Causes: haemolytic anaemia, malaria, G6PD deficiency, haemolytic disease of newborn.
2. Hepatic / Hepatocellular — liver cell injury → ↑ both conjugated + unconjugated. Causes: viral hepatitis, cirrhosis, drugs, Wilson's, Crigler-Najjar (UGT defect), Gilbert's (mild UGT reduction), Dubin-Johnson.
3. Post-hepatic / Obstructive — bile duct blockage → ↑ conjugated; pale stools + dark urine; pruritus. Causes: gallstones, pancreatic head Ca, cholangiocarcinoma, stricture.
Neonatal physiological jaundice — immature UGT; ↑ unconjugated; peaks day 3-5; resolves spontaneously; phototherapy for severe (UV converts bilirubin to lumirubin).

Structure of DNA:
• Double helix (Watson-Crick, 1953; Nobel 1962).
• Two antiparallel strands (5'→3' + 3'→5') coiled around a common axis.
• Backbone: alternating sugar (2'-deoxyribose) + phosphate; outside.
• Bases: inside; pairs by H-bonds (A-T: 2 H-bonds, G-C: 3 H-bonds). Purines (A, G, 2 rings) pair with pyrimidines (T, C, 1 ring). Chargaff's rule: A = T, G = C.
• Helix parameters: 10 base pairs per turn, 3.4 nm pitch, 2 nm diameter, right-handed (B-form).
• Major + minor grooves (protein binding sites).
• Forms: B-DNA (common), A-DNA (dehydrated), Z-DNA (left-handed).

Structure of RNA:
• Single-stranded (mostly); ribose + phosphate backbone; bases A, U, G, C.
• Secondary structure: hairpin, loop, stem.
3 main types:
• mRNA — ~5%; linear; 5' cap + 3' poly-A tail; codon-containing.
• tRNA — ~15%; cloverleaf shape (L-shape 3D); 76-90 nt; anticodon loop + CCA-3' end for amino acid; one tRNA per aa.
• rRNA — ~80%; structural + catalytic component of ribosome; 28S + 18S + 5.8S + 5S (eukaryotic).
Other: snRNA (splicing), miRNA (gene regulation), siRNA.

Semi-conservative Replication — Meselson-Stahl:
Each parental strand serves as template for new complementary strand; each daughter has 1 parent + 1 new strand (demonstrated by density-gradient centrifugation with ¹⁵N-labelled E.coli).

DNA Replication Steps:
1. Initiation:
• Origin of replication (OriC) — A-T rich; recognised by initiator proteins (DnaA in bacteria).
• Helicase 🔊 (DnaB) unwinds the double helix using ATP.
• Single-Strand Binding proteins (SSB) prevent re-annealing.
• Topoisomerase (gyrase) relieves super-coiling ahead of fork.
• Primase synthesises short RNA primer (~10 nt).
2. Elongation:
• DNA polymerase III (prokaryote) / pol δ + ε (eukaryote) adds dNTPs 5'→3' complementary to template.
• Leading strand synthesised continuously (3'→5' template).
• Lagging strand synthesised discontinuously in Okazaki fragments 🔊 (~100 – 200 nt in eukaryotes), whose primers are later removed.
• DNA polymerase I (prokaryote) / FEN-1 (eukaryote) removes RNA primer + fills gap with DNA.
• DNA ligase seals nicks.
3. Termination:
• Ter sites in prokaryotes; telomeres + telomerase in eukaryotes.
• Fidelity: polymerase proof-reading (3'→5' exonuclease); mismatch repair; error rate ~1 in 10⁹.

Transcription (DNA → mRNA):
Site: nucleus (eukaryotes); cytoplasm (prokaryotes).
Enzyme: RNA polymerase — 3 types in eukaryotes (I: rRNA; II: mRNA; III: tRNA + 5S rRNA).
Steps:
1. Initiation — RNA polymerase + transcription factors bind promoter (TATA box at -25 bp).
2. Elongation — polymerase reads template strand 3'→5'; synthesises mRNA 5'→3'; unwinds ahead, rewinds behind.
3. Termination — prokaryote: rho-dependent OR intrinsic (hairpin + poly-U); eukaryote: polyadenylation signal (AAUAAA).
Post-transcriptional modifications (eukaryotes):
• 5' capping with 7-methyl-guanosine.
• 3' polyadenylation (poly-A tail).
• Splicing (introns removed, exons joined by spliceosome containing snRNPs).
Inhibitors: Rifampicin (bacterial RNA pol — anti-TB); Actinomycin D (all polymerases — antineoplastic); α-amanitin (eukaryotic pol II — Amanita mushroom toxin).

Genetic Code:
• Triplet — 3 bases = 1 codon.
• 4³ = 64 codons; 61 code for 20 aa; 3 stop (UAA, UAG, UGA).
• AUG = start codon = Methionine.
• Universal (same in all organisms — few exceptions in mitochondria).
• Degenerate (one aa → multiple codons; wobble at 3rd base).
• Non-overlapping, comma-less.
• Read 5'→3' on mRNA.
Crick, Brenner, Khorana, Holley, Nirenberg deciphered it (Nobel 1968).

Translation (mRNA → Protein):
Site: ribosome (cytosol / RER).
Components: mRNA, aminoacyl-tRNA, ribosome (70S prokaryote; 80S eukaryote), GTP, initiation + elongation + release factors.
Stages:
1. Initiation:
• Small ribosomal subunit binds 5' mRNA cap (Kozak sequence in eukaryotes; Shine-Dalgarno in prokaryotes).
• Initiator tRNA (Met-tRNAi in eukaryotes; fMet-tRNA in prokaryotes) binds AUG in P site.
• Large subunit joins → complete ribosome with 3 sites: A (aminoacyl), P (peptidyl), E (exit).
2. Elongation:
Cycle of 3 steps:
(a) Aminoacyl-tRNA enters A site (GTP hydrolysis by EF-Tu).
(b) Peptide bond formed by peptidyl transferase (rRNA ribozyme) — growing chain shifts to A-site tRNA.
(c) Translocation (EF-G + GTP) — ribosome moves 3 nt along mRNA; tRNA shifts P → E → exits.
3. Termination:
Stop codon → release factors (RF-1, RF-2, RF-3) → polypeptide released from ribosome; ribosome dissociates.
Post-translational modifications: folding (chaperones), glycosylation, phosphorylation, cleavage (proinsulin → insulin), ubiquitination.
Inhibitors (antibiotics): Tetracycline (30S, A-site); Streptomycin (30S initiation); Chloramphenicol (50S, peptidyl transferase); Erythromycin (50S, translocation); Puromycin (premature termination; both 30S + 50S).

Biosynthesis of Purine Nucleotides (De Novo):
Site: cytosol; occurs in most cells (liver most active).
Key substrate: PRPP (5-phosphoribosyl-1-pyrophosphate) — activated ribose.
Atoms of the ring come from: N1 from Asp; C2 + C8 from formyl-THF; N3 + N9 from Gln; C4 + C5 + N7 from Gly; C6 from CO₂.
11-step pathway produces IMP (inosine monophosphate, hypoxanthine) first.
IMP → AMP (via adenylosuccinate) — needs GTP + Asp.
IMP → GMP (via XMP) — needs ATP + Gln + NAD⁺.
Rate-limiting: PRPP amidotransferase (regulated by AMP + GMP end-product inhibition).
Salvage pathway — reuse of free purines:
• HGPRT — hypoxanthine / guanine + PRPP → IMP / GMP.
• APRT — adenine + PRPP → AMP.
Deficiency of HGPRT → Lesch-Nyhan syndrome (X-linked; gout + self-mutilation + retardation in children).

Catabolism of Purines:
AMP → Adenosine → Inosine → Hypoxanthine → Xanthine → Uric acid (by xanthine oxidase).
GMP → Guanosine → Guanine → Xanthine → Uric acid.
Uric acid is the end product in humans + primates + birds + reptiles (lost uricase). Other mammals convert to allantoin.
Normal serum uric acid: ♂ 3.5–7.2 mg/dL; ♀ 2.6–6.0 mg/dL.

Hyperuricaemia & Gout:
Definition: Serum uric acid > 7 mg/dL (♂) / > 6 mg/dL (♀).
Causes:
• Overproduction — HGPRT deficiency (Lesch-Nyhan), ↑ PRPP synthetase, high purine diet (red meat, organ meats, sea food, beer), cancer chemotherapy (tumour lysis).
• Under-excretion — chronic kidney disease, diuretics (thiazide), alcohol, low-dose aspirin, acidosis.
Gout — clinical features:
• Acute attack: excruciating pain, redness, swelling of joint (classically 1st metatarsophalangeal joint = podagra), often at night.
• Chronic: tophi (uric acid deposits in ear, tendons, joints), uric acid kidney stones, nephropathy.
• Triggered by big meal, alcohol binge, trauma, surgery.
Diagnosis: serum uric acid; joint aspirate — negatively birefringent needle-shaped monosodium urate crystals under polarised light.
Treatment:
• Acute: NSAIDs, colchicine, glucocorticoids.
• Chronic / Prophylaxis:
 — Allopurinol / Febuxostat — xanthine oxidase inhibitors (↓ uric acid production).
 — Probenecid — uricosuric (↑ renal excretion).
 — Pegloticase / Rasburicase — recombinant uricase (converts to allantoin).
• Diet: avoid purine-rich foods + alcohol; hydrate well.

Definition & Properties:
Enzyme = biological catalyst (mostly protein; some RNA = ribozymes) that accelerates specific biochemical reactions by 10⁶-10¹² fold without being permanently altered.
Properties:
• Highly specific (substrate, reaction, stereochemistry).
• Catalytic — small amount catalyses large conversion.
• Protein in nature (mostly) → sensitive to pH, temperature, salts (denaturation).
• Lower activation energy (transition state stabilisation).
• Reversible — unless modified.
• Optimum activity at physiological pH (7.4) and 37 °C.
• Require cofactors (metal ion) / coenzymes (organic; often vitamins — NAD, FAD, CoA, PLP, TPP).
• Regulated (allosteric, covalent modification, inducers, repressors).
• Classified by IUB (Enzyme Commission).

IUB Classification (6 Classes):
Each enzyme given EC number (4 digits).

Class	Name	Reaction catalysed	Example
1	Oxidoreductases	Redox (transfer H, electrons, O)	Lactate dehydrogenase, catalase
2	Transferases	Transfer functional group (-NH₂, -CH₃, -PO₄)	Aminotransferases, kinases
3	Hydrolases	Hydrolysis of bond (ester, peptide, glycosidic)	Lipase, amylase, pepsin, trypsin
4	Lyases	Addition / removal without hydrolysis (C=C / C-O)	Aldolase, fumarase, decarboxylase
5	Isomerases	Intramolecular rearrangement	Phosphoglucose isomerase, mutase
6	Ligases (Synthetases)	Join two molecules with ATP hydrolysis	DNA ligase, pyruvate carboxylase

Mnemonic: "Over The Hill Lies Isolated Ligase".

Michaelis-Menten Kinetics:
E + S ⇌ ES → E + P.
V₀ = V_max × [S] / (K_m + [S])
where:
• V_max = maximum velocity (when all enzyme saturated).
• K_m (Michaelis constant) = [S] at which V = ½ V_max; measure of enzyme-substrate affinity (low Km → high affinity).
Curve is hyperbolic.
At low [S]: V ∝ [S] (first order).
At high [S]: V → V_max (zero order).
Turnover number (k_cat) = V_max/[E]_total.

Lineweaver-Burk (Double-Reciprocal) Plot:
Taking reciprocal of Michaelis-Menten equation linearises it:
1/V₀ = (K_m/V_max) × (1/[S]) + 1/V_max
Plot of 1/V vs 1/[S] gives straight line.
• Y-intercept = 1/V_max.
• X-intercept = −1/K_m.
• Slope = K_m/V_max.
Use: precisely determine V_max + K_m; distinguish types of inhibition.

Classification of Inhibitors:
1. Reversible — bind non-covalently; reversed by removal.
  • Competitive
  • Non-competitive
  • Uncompetitive
  • Mixed
2. Irreversible — covalent modification of enzyme.
3. Allosteric inhibitors — bind at site other than active site → conformational change.
4. Suicide inhibitors — converted by enzyme to reactive species that covalently inactivate the enzyme.

1. Competitive Inhibition:
Inhibitor structurally resembles substrate → competes for active site.
Effect: K_m ↑ (apparent); V_max unchanged. Overcome by ↑ [S].
Lineweaver-Burk: lines intersect on Y-axis.
Examples: Malonate vs succinate (succinate dehydrogenase); Methotrexate vs folate (dihydrofolate reductase); Allopurinol vs hypoxanthine (xanthine oxidase); Statins vs HMG-CoA reductase substrate.

2. Non-Competitive Inhibition:
Inhibitor binds at a site different from active site (allosteric); does not compete with substrate.
Effect: V_max ↓; K_m unchanged.
Lineweaver-Burk: lines intersect on X-axis.
Examples: Heavy metals (Pb, Hg) on thiol enzymes; Cyanide on cytochrome oxidase (though some say uncompetitive).

3. Uncompetitive Inhibition:
Inhibitor binds only to ES complex (not free enzyme).
Effect: both V_max + K_m decrease.
Lineweaver-Burk: parallel lines.
Example: Lithium on inositol monophosphatase.

4. Irreversible Inhibition:
Covalent modification of active-site residue.
Examples: Aspirin (acetylates COX); Omeprazole (covalent disulphide with H⁺/K⁺ ATPase); Penicillin (acylates transpeptidase); Diisopropyl fluorophosphate (DFP) inhibits AChE; Organophosphates (AChE).

Regulation of Enzymes:
1. Allosteric regulation — effector binds regulatory site → conformational change → altered activity; sigmoidal kinetics.
Example: PFK-1 inhibited by ATP/citrate, activated by AMP/F-2,6-BP.
2. Covalent modification — phosphorylation (kinase), dephosphorylation (phosphatase); acetylation; methylation; ubiquitination.
Example: Glycogen phosphorylase activated by phosphorylation; glycogen synthase inhibited by phosphorylation.
3. Proteolytic activation (zymogens) — inactive pro-enzyme cleaved to active form.
Example: Pepsinogen → pepsin; trypsinogen → trypsin; prothrombin → thrombin.
4. Enzyme induction + repression (transcriptional) — hormone / metabolite alters gene expression.
Example: Insulin induces glucokinase; cortisol induces gluconeogenic enzymes.
5. Feedback inhibition — end product inhibits the first enzyme of pathway.
Example: ATP inhibits PFK-1; CTP inhibits aspartate transcarbamoylase (pyrimidine synthesis).
6. Compartmentalisation — different enzymes in different organelles.
7. Isoenzymes — different forms in different tissues with different regulation.

Therapeutic Uses of Enzymes:
• Streptokinase + Urokinase + Tissue plasminogen activator (tPA) — fibrinolytic drugs for MI, stroke, pulmonary embolism (activate plasminogen → plasmin → dissolve clot).
• Pancreatin (amylase + lipase + protease) — pancreatic enzyme replacement in chronic pancreatitis, cystic fibrosis.
• L-asparaginase — depletes circulating asparagine → treats acute lymphoblastic leukaemia (ALL).
• Uricase (rasburicase) — converts uric acid → allantoin; gout + tumour lysis.
• Collagenase — topical debridement of wounds.
• Hyaluronidase — facilitates absorption of IM / SC injections (spreading factor).
• Pepsin + Papain — digestive aids.
• Lactase — lactose intolerance.
• Serratiopeptidase, Bromelain, Trypsin-Chymotrypsin — anti-inflammatory.
• Recombinant enzymes — Glucocerebrosidase (Gaucher's), α-galactosidase (Fabry's), idursulfase (Hunter syndrome).

Diagnostic Uses — Serum Enzyme Markers:

Condition	Marker enzyme
Myocardial infarction	Troponin (not enzyme); CK-MB, LDH-1 (> LDH-2 flip), AST
Liver disease (hepatitis)	ALT > AST (ratio >1 in viral); ALP + GGT (cholestasis); bilirubin
Alcoholic liver	AST / ALT > 2; GGT ↑
Acute pancreatitis	Amylase (↑ 3× within 12 h); Lipase (more specific, stays ↑ 14 d)
Bone disease (Paget's, osteoblastic metastasis)	Alkaline phosphatase (ALP)
Prostate carcinoma	Acid phosphatase; PSA
Muscle injury / Duchenne	Creatine kinase (CK-MM)
Obstructive jaundice	ALP ↑↑, GGT ↑, direct bilirubin ↑

Isoenzymes:
Multiple forms of same enzyme; differ in aa sequence + tissue distribution.
LDH (Lactate dehydrogenase) — 5 isoforms:
• LDH-1 (H4) — heart, RBC.
• LDH-2 (H3M) — heart, RES.
• LDH-3 (H2M2) — brain, kidney, lung.
• LDH-4 (HM3) — liver, muscle.
• LDH-5 (M4) — liver, skeletal muscle.
Normal: LDH-2 > LDH-1. In MI, LDH-1 > LDH-2 "flip".
CK (Creatine kinase) — 3 isoforms:
• CK-MM — skeletal muscle.
• CK-MB — heart (↑ in MI).
• CK-BB — brain.
ALP — bone + liver + intestinal + placental.

Common Coenzymes (with Vitamin source):

Coenzyme	Vitamin source	Function
TPP	B1 (Thiamine)	Decarboxylation (PDH, α-KG DH, transketolase)
FAD / FMN	B2 (Riboflavin)	Redox (succinate DH, ETC)
NAD / NADP	B3 (Niacin)	Redox (dehydrogenases)
CoA	B5 (Pantothenic acid)	Acyl transfer (acetyl-CoA)
PLP	B6 (Pyridoxine)	Transamination + decarboxylation
Biotin	B7	Carboxylation (pyruvate carboxylase)
THF	B9 (Folic acid)	1C transfer
Methylcobalamin	B12	Methyl transfer; homocysteine → methionine
Ascorbate	C	Collagen hydroxylation

Glycogenesis (synthesis):
• Site: cytosol of liver + muscle.
• Glucose → G-6-P → G-1-P → UDP-glucose → glycogen (α-1,4 + α-1,6 branches).
• Key enzymes: glycogen synthase + branching enzyme.
• Activated by insulin (dephosphorylated form active).
Glycogenolysis (breakdown):
• Glycogen → G-1-P (by glycogen phosphorylase — breaks α-1,4; debranching enzyme breaks α-1,6) → G-6-P → glucose (liver only, via G-6-Pase) OR enters glycolysis (muscle).
• Activated by glucagon (liver) + adrenaline (both); phosphorylated form active.

Glycogen Storage Diseases:

Type	Name	Enzyme Defect	Organ	Features
I	Von Gierke's	Glucose-6-Phosphatase	Liver, kidney	Severe fasting hypoglycaemia, hepatomegaly, lactic acidosis
II	Pompe's	Lysosomal α-1,4-glucosidase	All tissues (lysosome)	Cardiomegaly, hypotonia, infant death
III	Cori's	Debranching enzyme	Liver + muscle	Mild — hypoglycaemia
IV	Andersen's	Branching enzyme	Liver	Cirrhosis
V	McArdle's	Muscle phosphorylase	Muscle	Exercise intolerance, muscle cramps, myoglobinuria
VI	Hers'	Liver phosphorylase	Liver	Mild hypoglycaemia

Biological Significance of Cholesterol:
• Structural: component of cell membranes → fluidity modulator.
• Precursor of bile acids (cholic + chenodeoxycholic) → fat absorption.
• Precursor of steroid hormones — progesterone, aldosterone, cortisol, testosterone, estrogen.
• Precursor of Vitamin D (7-dehydrocholesterol + UV → cholecalciferol).
• Myelin sheath lipid.
Synthesis: from acetyl-CoA; rate-limiting enzyme HMG-CoA reductase (target of statins); produces ~1 g/day in liver.

Disorders of Lipid Metabolism:
1. Hypercholesterolaemia — total chol > 240 mg/dL.
• Familial (AD): LDL-receptor defect → very high LDL; early MI.
• Acquired: high saturated fat, obesity, DM, hypothyroidism.
• Classification: total chol, LDL ("bad"), HDL ("good"), triglycerides.
2. Atherosclerosis — plaque build-up in arterial wall.
Sequence: endothelial injury → LDL oxidation → macrophage uptake → foam cells → fatty streak → fibrous plaque → stenosis + rupture → thrombosis → MI / stroke.
Risk factors: ↑ LDL, ↓ HDL, HTN, DM, smoking, obesity, age, family history.
3. Fatty liver (hepatic steatosis) — TG accumulation in hepatocytes; causes: obesity, alcohol, insulin resistance, malnutrition (choline / Met deficiency).
4. Obesity — BMI > 30; central (visceral) more dangerous; metabolic syndrome.
Treatment:
• Lifestyle — diet, exercise, weight loss.
• Statins (atorvastatin, rosuvastatin) — HMG-CoA reductase inhibitors; lower LDL 20-60 %.
• Ezetimibe — blocks intestinal chol absorption.
• Bile acid sequestrants (cholestyramine).
• Fibrates (TG ↓); Nicotinic acid (HDL ↑).
• PCSK9 inhibitors (evolocumab, alirocumab) — severe familial.