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Biochemistry LAQs - Paper 1 - Simple Answers

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1. VITAMIN D METABOLISM

1. Endogenous Synthesis in Skin

• Sunlight (UV-B) hits the skin and converts a cholesterol-like compound (7-dehydrocholesterol) into Vitamin D3 (cholecalciferol).
• Think of it as: Sun + skin cholesterol = Vitamin D3

2. Sequential Hydroxylations (Making the Active Form)

• Calcitriol is the fully active hormone.
• Low calcium or PTH stimulates the kidney to make more calcitriol.

3. Role in Calcium Homeostasis

1. Gut - increases calcium absorption from food
2. Bone - releases calcium from bones (with PTH)
3. Kidney - reduces calcium loss in urine

4. Deficiency Disorders

• Children → Rickets: soft, deformed bones; bow legs, knock knees, "rachitic rosary" on ribs
• Adults → Osteomalacia: soft bones, bone pain, muscle weakness
• Elderly/severe → Osteoporosis (combined with other factors)
• Cause of deficiency: lack of sunlight, poor diet, kidney disease (can't make calcitriol), liver disease

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2. FATTY ACIDS

1. Activation (Turning Fat into a Usable Form)

• Fatty acids must be "activated" before they can be burned.
• They combine with CoA (coenzyme A) to form Fatty acyl-CoA.
• This happens in the cytoplasm (cytosol).
• Enzyme: Acyl-CoA synthetase (also called thiokinase)
• Costs 2 ATP (ATP → AMP, so equivalent to 2 ATP lost)

2. Mitochondrial Transport - The Carnitine Shuttle

• Fatty acyl-CoA is too big to cross the mitochondrial inner membrane on its own.
• Carnitine acts as a "ferry" or "taxi" to carry it across.

1. Fatty acyl-CoA + Carnitine → Acylcarnitine (enzyme: CPT-1, on outer membrane)
2. Acylcarnitine crosses the inner membrane via a transporter
3. Inside: Acylcarnitine → Fatty acyl-CoA + Carnitine released (enzyme: CPT-2)
4. Carnitine ferries back out

• CPT-1 is the rate-limiting step and is inhibited by malonyl-CoA (so when you're making fat, you don't burn it at the same time - smart!)

3. Reactions of the Spiral (Beta-oxidation) Pathway

4. Energy from Palmitic Acid (16 carbons)

• Palmitic acid (C16) goes through 7 rounds of beta-oxidation
• Products: 8 Acetyl-CoA + 7 FADH₂ + 7 NADH

• 8 Acetyl-CoA × 10 ATP = 80 ATP
• 7 FADH₂ × 1.5 ATP = 10.5 ATP
• 7 NADH × 2.5 ATP = 17.5 ATP
• Total = 108 ATP
• Subtract 2 ATP for activation = Net 106 ATP

5. Associated Metabolic Blocks

• Carnitine deficiency: Can't transport fatty acids into mitochondria → fat accumulates, muscle weakness, hypoglycemia
• CPT-1 deficiency: Same result as carnitine deficiency
• MCAD deficiency (medium-chain acyl-CoA dehydrogenase): Can't oxidize medium-chain fats; dangerous in fasting - hypoglycemia, sudden death in children
• Refsum disease: Can't oxidize branched-chain fatty acids → nerve damage

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3. VITAMIN B12

1 & 2. Absorption and Transport (Intrinsic Factor)

• B12 from food binds to R-protein (haptocorrin) in saliva first.
• In the stomach, Intrinsic Factor (IF) is made by parietal cells.
• In the small intestine, R-protein is digested off and B12 binds to IF.
• The B12-IF complex is absorbed in the terminal ileum (last part of small intestine) via specific receptors (cubilin).
• In blood, B12 is carried by proteins called transcobalamin I, II, III.
• Transcobalamin II is the most important for delivery to cells.

3. Biochemical Role as a Coenzyme

• Converts homocysteine → methionine
• Enzyme: methionine synthase
• Also regenerates THF (tetrahydrofolate) from methyl-THF
• This is the link between B12 and folate!

• Converts methylmalonyl-CoA → succinyl-CoA
• Enzyme: methylmalonyl-CoA mutase
• Important for odd-chain fatty acid oxidation
• Deficiency → methylmalonic acid builds up in blood/urine (diagnostic marker)

4. Neurological Manifestations

• Subacute combined degeneration of spinal cord - affects both sensory and motor tracts
• Symptoms: Tingling/numbness in hands & feet (peripheral neuropathy), balance problems, difficulty walking, dementia, depression, memory loss
• Remember: folate deficiency gives anemia but NOT the neurological damage

5. The Folate Trap

• B12 is needed to convert methyl-THF → THF
• Without B12, all folate gets "trapped" as methyl-THF (a dead-end form)
• THF is needed to make DNA building blocks
• So B12 deficiency → effectively traps folate → can't make DNA → megaloblastic anemia (same as folate deficiency anemia)
• This is why giving folate to a B12-deficient patient fixes the blood but doesn't fix the nerve damage - a dangerous situation!

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4. CHOLESTEROL BIOSYNTHESIS

1. Intracellular Pathway from Acetyl-CoA

1. 2 Acetyl-CoA → Acetoacetyl-CoA
2. + 1 more Acetyl-CoA → HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) - in cytosol
3. HMG-CoA → Mevalonate ← RATE-LIMITING STEP (enzyme: HMG-CoA reductase)
4. Mevalonate → Isoprene units (IPP)
5. 6 Isoprene units → Squalene (30C)
6. Squalene → Lanosterol → Cholesterol (27C)

2. Rate-Limiting Step: HMG-CoA Reductase

• This enzyme converts HMG-CoA → Mevalonate
• It is the bottleneck of the whole pathway
• Statins (e.g., atorvastatin, lovastatin) BLOCK this enzyme → less cholesterol made

3. Feedback Regulation

• High cholesterol → SREBP (a transcription factor) is kept inactive → less HMG-CoA reductase made
• Low cholesterol → SREBP becomes active → more HMG-CoA reductase made → more cholesterol
• Insulin activates HMG-CoA reductase (fed state = make cholesterol)
• Glucagon and cortisol inhibit it (fasting = save energy)
• Cholesterol also reduces LDL receptor expression (so less uptake from blood when already plenty)

4. Products Derived from Cholesterol

• Bile acids/salts - for fat digestion
• Steroid hormones - cortisol, aldosterone, sex hormones (estrogen, testosterone, progesterone)
• Vitamin D - made from cholesterol in skin
• Cell membranes - cholesterol gives rigidity
• Myelin in nerves

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5. GLYCOLYSIS

1. Complete Pathways

1. Glucose + ATP → Glucose-6-phosphate (enzyme: hexokinase/glucokinase)
2. G-6-P → Fructose-6-phosphate
3. F-6-P + ATP → Fructose-1,6-bisphosphate (PFK-1 - RATE LIMITING STEP)
4. F-1,6-BP → 2× DHAP + G3P
5. DHAP → G3P (so now 2× G3P)
6. G3P → 1,3-BPG (+ 2 NADH made)
7. 1,3-BPG → 3-phosphoglycerate (+ 2 ATP by substrate-level phosphorylation)
8. 3-PG → 2-phosphoglycerate
9. 2-PG → Phosphoenolpyruvate (PEP)
10. PEP → Pyruvate (+ 2 ATP) - enzyme: pyruvate kinase

• Pyruvate → Acetyl-CoA (pyruvate dehydrogenase) → TCA cycle → lots of ATP

• Pyruvate → Lactate (lactate dehydrogenase)
• This regenerates NAD+ so glycolysis can keep going
• Net: 2 ATP per glucose (no oxygen)

2. Rate-Limiting Steps

• Hexokinase/Glucokinase (Step 1)
• PFK-1 (Step 3) - THE MOST IMPORTANT rate-limiting step
• Pyruvate kinase (Step 10)

3. Hormonal Regulation

• PFK-1 is activated by: AMP, ADP, F-2,6-BP (low energy signal → make energy)
• PFK-1 is inhibited by: ATP, citrate (high energy signal → no need to make more)

4. Energetics

5. Rapoport-Luebering Shunt

• Normally: 1,3-BPG → 3-PG (makes ATP)
• In the shunt: 1,3-BPG → 2,3-BPG → 3-PG (NO ATP made!)
• Why is it important? 2,3-BPG binds to hemoglobin and reduces its affinity for oxygen → hemoglobin releases O₂ more easily to tissues
• Especially important in RBCs (which cannot make ATP by any other means)
• Clinical: stored blood has low 2,3-BPG → Hb holds O₂ tightly → tissues don't get enough O₂ (transfusion problem)

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6. TCA CYCLE (Krebs Cycle / Citric Acid Cycle)

1. Detailed Steps

2. Regulatory Checkpoints

3. Energetics (ATP yield per Acetyl-CoA)

• 3 NADH × 2.5 ATP = 7.5 ATP
• 1 FADH₂ × 1.5 ATP = 1.5 ATP
• 1 GTP = 1 ATP
• Total = 10 ATP per Acetyl-CoA

4. Amphibolic and Anaplerotic Roles

• Breaking down: burns acetyl-CoA for energy (catabolism)
• Building up: provides building blocks for making amino acids, fatty acids, glucose (anabolism)

• Most important: Pyruvate carboxylase converts pyruvate → OAA (replenishes OAA)
• Also: amino acids feed in (e.g., glutamate → α-ketoglutarate)
• Why needed: if OAA runs low, the cycle slows down even if Acetyl-CoA is available

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7. ENZYME INHIBITION

1. Competitive vs Non-Competitive Inhibition

• Competitive: inhibitor and substrate fight for the same seat. More substrate = inhibitor loses.
• Non-competitive: inhibitor sits somewhere else and slows the enzyme down regardless.

2. Lineweaver-Burk Plots (Double Reciprocal Plot)

• X-intercept = -1/Km
• Y-intercept = 1/Vmax

• Lines cross on Y-axis (same Vmax)
• X-intercept shifts (different Km - higher Km with inhibitor)

• Lines cross on X-axis (same Km)
• Y-intercept shifts up (lower Vmax)

3. Changes in Km and Vmax

4. Clinical Examples

• Competitive inhibitor of HMG-CoA reductase
• Blocks cholesterol synthesis
• Used for: high cholesterol, preventing heart attacks

• Irreversible (suicide) inhibitor of COX-1 and COX-2 enzymes
• Blocks prostaglandin and thromboxane synthesis
• Effect: anti-platelet, anti-inflammatory, analgesic
• Irreversible means even adding more arachidonic acid won't help (different from competitive)

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8. OXIDATIVE PHOSPHORYLATION

1. The Mitochondrial Respiratory Chain (Complexes I-V)

2. Chemiosmotic Theory (Mitchell's Theory)

1. Complexes I, III, IV pump H⁺ (protons) OUT of the mitochondrial matrix → intermembrane space
2. This creates a proton gradient (electrochemical gradient) - like a battery
3. H⁺ wants to flow back in - can only do so through Complex V (ATP synthase)
4. H⁺ flowing back through Complex V drives ATP synthesis
5. This is called chemiosmosis - chemical + osmosis (concentration gradient)

3. Inhibitors and Uncouplers

• Rotenone (insecticide) - blocks Complex I
• Antimycin A - blocks Complex III
• Cyanide (CN⁻), CO, Azide - block Complex IV → death (no ATP made, O₂ not used)

• Oligomycin - directly blocks Complex V → no ATP made

• H⁺ leaks back WITHOUT going through ATP synthase
• Gradient is destroyed → no ATP made
• BUT electrons still flow → energy released as HEAT instead
• Example: 2,4-DNP (dinitrophenol) - once used as a diet pill (dangerous!)
• Example: Thermogenin (UCP-1) in brown fat - natural uncoupler that generates heat in newborns/hibernating animals

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9. PROTEIN BIOSYNTHESIS (Translation)

1. Phases of Translation in Prokaryotes

1. Small ribosome subunit (30S) binds to mRNA at the Shine-Dalgarno sequence
2. Initiator tRNA (fMet-tRNA) comes in - carrying formyl-methionine (fMet)
3. Large subunit (50S) joins → complete 70S ribosome
4. Needs: IF1, IF2, IF3 (initiation factors) + GTP

1. Aminoacyl-tRNA enters the A site (with EF-Tu + GTP)
2. Peptide bond formed between growing chain (P site) and new amino acid (A site)
3. Enzyme: peptidyl transferase (part of 23S rRNA - a ribozyme!)
4. Ribosome moves one codon forward (translocation) - needs EF-G + GTP
5. tRNA moves: A→P→E (Exit site)

1. A stop codon (UAA, UAG, UGA) enters the A site
2. No tRNA fits - instead release factors (RF1, RF2) bind
3. Polypeptide chain released
4. Ribosome dissociates

2. Initiation and Elongation Factors

3. Codon-Anticodon Recognition and Wobble Hypothesis

• Each codon (3 bases on mRNA) is read by an anticodon (3 bases on tRNA)
• Positions 1 & 2 of codon must match EXACTLY
• Position 3 (wobble position) can be flexible - one tRNA can read multiple codons!

• Inosine (I) in tRNA anticodon can pair with U, C, or A in mRNA
• This means we need FEWER tRNA types than codons (only ~45 tRNA for 61 codons)

4. Post-Translational Modifications

• Glycosylation - sugars added (in ER/Golgi) → glycoproteins
• Phosphorylation - phosphate group added → activates/deactivates proteins (signaling)
• Methylation, acetylation - common for histone proteins
• Hydroxylation - proline → hydroxyproline in collagen (needs Vitamin C!)
• Signal peptide cleavage - signal sequence cut off after protein reaches destination
• Disulfide bond formation - stabilizes protein structure (in ER)
• Ubiquitination - tags protein for destruction

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10. ACID-BASE REGULATION

1. Buffer Mechanisms

• Lungs control CO₂
• Too acidic → breathe faster → blow off CO₂ → pH rises
• Too alkaline → breathe slower → retain CO₂ → pH falls
• CO₂ + H₂O ↔ H₂CO₃ ↔ H⁺ + HCO₃⁻

• Kidneys control HCO₃⁻
• Too acidic → kidneys absorb more HCO₃⁻, excrete more H⁺ (as NH₄⁺ and H₂PO₄⁻)
• Too alkaline → kidneys excrete more HCO₃⁻, retain H⁺
• Bicarbonate buffer is the most important buffer in blood

• Hemoglobin (in RBCs) - buffers CO₂ in blood
• Phosphate buffer - mainly in urine and cells
• Protein buffer - mainly inside cells

2. Henderson-Hasselbalch Equation

• pH = 7.4
• HCO₃⁻ = 24 mEq/L
• pCO₂ = 40 mmHg
• Ratio = 20:1 (HCO₃⁻:CO₂)

• High CO₂ → acid (pH falls)
• High HCO₃⁻ → alkaline (pH rises)

3. Compensatory Responses

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11. BILIRUBIN METABOLISM

1. Complete Pathway of Heme Catabolism

• Old RBCs are broken down
• Hemoglobin → Heme + Globin
• Heme → Biliverdin (green) - enzyme: heme oxygenase
• Biliverdin → Bilirubin (yellow/orange) - enzyme: biliverdin reductase
• At this stage, bilirubin is unconjugated (indirect) bilirubin - fat-soluble, not water-soluble

2. Transport in Blood

• Unconjugated bilirubin is fat-soluble → can't dissolve in blood alone
• It binds to albumin for transport to the liver
• It is NOT in urine at this stage (albumin-bound, too large)

3. Hepatic Conjugation

• In the liver, bilirubin is taken up from albumin
• It is conjugated (attached) to glucuronic acid by the enzyme UDP-glucuronosyltransferase (UGT)
• Now it is conjugated (direct) bilirubin - water-soluble
• Secreted into bile

4. Intestinal Excretion

• Conjugated bilirubin enters the gut via bile
• Gut bacteria convert it to urobilinogen
• Most urobilinogen → stercobilin (brown color of stool)
• Small amount of urobilinogen absorbed back into blood → some excreted in urine as urobilin (yellow color of urine)

5. Differential Diagnosis of Jaundice (Yellow skin/eyes)

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12. UREA CYCLE

1. Detailed Enzymatic Steps

1. NH₃ + CO₂ + 2ATP → Carbamoyl phosphate (enzyme: CPS-1 - carbamoyl phosphate synthetase I)
2. Carbamoyl phosphate + Ornithine → Citrulline (enzyme: OTC - ornithine transcarbamoylase)
   • Citrulline exits to the cytosol

• Ornithine goes back into mitochondria to repeat the cycle
• Urea is excreted by kidneys

2. Biochemical Regulation

• N-acetylglutamate (NAG) activates CPS-I (the first enzyme)
• NAG is made when amino acids are abundant → stimulates urea cycle
• NAG synthase is activated by arginine → positive feedback (more arginine = more urea cycle activity)
• High protein diet → more amino acid breakdown → more ammonia → more urea cycle activity

3. Connection with TCA Cycle - Krebs Bicycle

• In Step 4, fumarate is released in the cytosol
• Fumarate enters the TCA cycle → becomes malate → OAA → aspartate
• Aspartate comes back to feed Step 3 of the urea cycle again
• This creates a "bicycle" (two linked cycles):
  • Urea cycle provides fumarate to TCA cycle
  • TCA cycle provides aspartate back to urea cycle
  • They share fumarate and aspartate as the connecting metabolites

4. Hyperammonemia and Urea Cycle Defects

• Cerebral edema (brain swelling)
• Vomiting, asterixis (liver flap - tremor)
• Confusion, coma, death

• NH₃ + α-ketoglutarate → glutamate → glutamine
• This depletes α-ketoglutarate from TCA cycle → TCA slows → less ATP → brain cells die
• Also causes cerebral edema

• Low protein diet
• Lactulose (reduces ammonia absorption from gut)
• Sodium benzoate/phenylbutyrate (alternative pathways to excrete nitrogen)
• Liver transplant in severe genetic cases

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